Semi-synthetic terephthalic acid via microorganisms that produce muconic acid

ABSTRACT

The invention provides a non-naturally occurring microbial organism having a muconate pathway having at least one exogenous nucleic acid encoding a muconate pathway enzyme expressed in a sufficient amount to produce muconate. The muconate pathway including an enzyme selected from the group consisting of a beta-ketothiolase, a beta-ketoadipyl-CoA hydrolase, a beta-ketoadipyl-CoA transferase, a beta-ketoadipyl-CoA ligase, a 2-fumarylacetate reductase, a 2-fumarylacetate dehydrogenase, a trans-3-hydroxy-4-hexendioate dehydratase, a 2-fumarylacetate aminotransferase, a 2-fumarylacetate aminating oxidoreductase, a trans-3-amino-4-hexenoate deaminase, a beta-ketoadipate enol-lactone hydrolase, a muconolactone isomerase, a muconate cycloisomerase, a beta-ketoadipyl-CoA dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA transferase, a 2,3-dehydroadipyl-CoA hydrolase, a 2,3-dehydroadipyl-CoA ligase, a muconate reductase, a 2-maleylacetate reductase, a 2-maleylacetate dehydrogenase, a cis-3-hydroxy-4-hexendioate dehydratase, a 2-maleylacetate aminoatransferase, a 2-maleylacetate aminating oxidoreductase, a cis-3-amino-4-hexendioate deaminase, and a muconate cis/trans isomerase. Other muconate pathway enzymes also are provided. Additionally provided are methods of producing muconate.

This application is a continuation of U.S. patent application Ser. No.12/851,478, filed Aug. 5, 2010, which claims the benefit of priority ofU.S. Provisional Application No. 61/231,637, filed Aug. 5, 2009, theentire contents of which are incorporated herein by this reference.Also, the entire contents of the ASCII text file entitled“GN00007US2_Sequence Listing.txt” created on Jun. 18, 2014, having asize of 34 kilobytes, is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present disclosure relates generally to the design of engineeredorganisms and, more specifically to organisms having selected genotypesfor the production of muconic acid.

Terephthalate (also known as terephthalic acid and PTA) is the immediateprecursor of polyethylene terephthalate (PET), used to make clothing,resins, plastic bottles and even as a poultry feed additive. Nearly allPTA is produced from para-xylene by the oxidation in air in a processknown as the Mid Century Process (Roffia et al., Ind. Eng. Chem. Prod.Res. Dev. 23:629-634 (1984)). This oxidation is conducted at hightemperature in an acetic acid solvent with a catalyst composed of cobaltand/or manganese salts. Para-xylene is derived from petrochemicalsources, and is formed by high severity catalytic reforming of naphtha.Xylene is also obtained from the pyrolysis gasoline stream in a naphthasteam cracker and by toluene disproportion.

PTA, toluene and other aromatic precursors are naturally degraded bysome bacteria. However, these degradation pathways typically involvemonooxygenases that operate irreversibly in the degradative direction.Hence, biosynthetic pathways for PTA are severely limited by theproperties of known enzymes to date.

Muconate (also known as muconic acid, MA) is an unsaturated dicarboxylicacid used as a raw material for resins, pharmaceuticals andagrochemicals. Muconate can be converted to adipic acid, a precursor ofNylon-6,6, via hydrogenation (Draths and Frost, J. Am. Chem. Soc. 116;399-400 (1994)). Alternately, muconate can be hydrogenated usingbiometallic nanocatalysts (Thomas et al., Chem. Commun. 10:1126-1127(2003)).

Muconate is a common degradation product of diverse aromatic compoundsin microbes. Several biocatalytic strategies for making cis,cis-muconatehave been developed. Engineered E. coli strains producing muconate fromglucose via shikimate pathway enzymes have been developed in the Frostlab (U.S. Pat. No. 5,487,987 (1996); Niu et al., Biotechnol Prog.,18:201-211 (2002)). These strains are able to produce 36.8 g/L of cis,cis-muconate after 48 hours of culturing under fed-batch fermenterconditions (22% of the maximum theoretical yield from glucose). Muconatehas also been produced biocatalytically from aromatic starting materialssuch as toluene, benzoic acid and catechol. Strains producing muconatefrom benzoate achieved titers of 13.5 g/L and productivity of 5.5 g/L/hr(Choi et al., J. Ferment. Bioeng. 84:70-76 (1997)). Muconate has alsobeen generated from the effluents of a styrene monomer production plant(Wu et al., Enzyme and Microbiology Technology 35:598-604 (2004)).

All biocatalytic pathways identified to date proceed through enzymes inthe shikimate pathway, or degradation enzymes from catechol.Consequently, they are limited to producing the cis, cis isomer ofmuconate, since these pathways involve ring-opening chemistry. Thedevelopment of pathways for producing trans, trans-muconate andcis,trans-muconate would be useful because these isomers can serve asdirect synthetic intermediates for producing renewable PTA via theinverse electron demand Diels-Alder reaction with acetylene. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides a non-naturally occurring microbial organismhaving a muconate pathway having at least one exogenous nucleic acidencoding a muconate pathway enzyme expressed in a sufficient amount toproduce muconate. The muconate pathway including an enzyme selected fromthe group consisting of a beta-ketothiolase, a beta-ketoadipyl-CoAhydrolase, a beta-ketoadipyl-CoA transferase, a beta-ketoadipyl-CoAligase, a 2-fumarylacetate reductase, a 2-fumarylacetate dehydrogenase,a trans-3-hydroxy-4-hexendioate dehydratase, a 2-fumarylacetateaminotransferase, a 2-fumarylacetate aminating oxidoreductase, atrans-3-amino-4-hexenoate deaminase, a beta-ketoadipate enol-lactonehydrolase, a muconolactone isomerase, a muconate cycloisomerase, abeta-ketoadipyl-CoA dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a2,3-dehydroadipyl-CoA transferase, a 2,3-dehydroadipyl-CoA hydrolase, a2,3-dehydroadipyl-CoA ligase, a muconate reductase, a 2-maleylacetatereductase, a 2-maleylacetate dehydrogenase, acis-3-hydroxy-4-hexendioate dehydratase, a 2-maleylacetateaminoatransferase, a 2-maleylacetate aminating oxidoreductase, acis-3-amino-4-hexendioate deaminase, and a muconate cis/trans isomerase.Other muconate pathway enzymes also are provided. Additionally providedare methods of producing muconate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of terepthalate from muconate and acetylenevia Diels-Alder chemistry. P1 is cyclohexa-2,5-diene-1,4-dicarboxylate.

FIG. 2 shows pathways to trans-trans muconate from succinyl CoA. Enzymesare A) beta-ketothiolase, B) beta-ketoadipyl-CoA hydrolase, transferaseand/or ligase, C) 2-fumarylacetate reductase, D) 2-fumarylacetatedehydrogenase, E) trans-3-hydroxy-4-hexendioate dehydratase, F)2-fumarylacetate aminotransferase and/or 2-fumarylacetate aminatingoxidoreductase, G) trans-3-amino-4-hexenoate deaminase, H)beta-ketoadipate enol-lactone hydrolase, I) muconolactone isomerase, J)muconate cycloisomerase, K) beta-ketoadipyl-CoA dehydrogenase, L)3-hydroxyadipyl-CoA dehydratase, M) 2,3-dehydroadipyl-CoA transferase,hydrolase or ligase, N) muconate reductase, O) 2-maleylacetatereductase, P) 2-maleylacetate dehydrogenase, Q)cis-3-hydroxy-4-hexendioate dehydratase, R) 2-maleylacetateaminotransferase and/or 2-maleylacetate aminating oxidoreductase, S)cis-3-amino-4-hexendioate deaminase, T) muconate cis/trans isomerase, W)muconate cis/trans isomerase.

FIG. 3 shows pathways to muconate from pyruvate and malonatesemialdehyde. Enzymes are A) 4-hydroxy-2-ketovalerate aldolase, B)2-oxopentenoate hydratase, C) 4-oxalocrotonate dehydrogenase, D)2-hydroxy-4-hexenedioate dehydratase, E) 4-hydroxy-2-oxohexanedioateoxidoreductase, F) 2,4-dihydroxyadipate dehydratase (acting on2-hydroxy), G) 2,4-dihydroxyadipate dehydratase (acting on 4-hydroxylgroup) and H) 3-hydroxy-4-hexenedioate dehydratase.

FIG. 4 shows pathways to muconate from pyruvate and succinicsemialdehyde. Enzymes are A) HODH aldolase, B) OHED hydratase, C) OHEDdecarboxylase, D) HODH formate-lyase and/or HODH dehydrogenase, E) OHEDformate-lyase and/or OHED dehydrogenase, F) 6-OHE dehydrogenase, G)3-hydroxyadipyl-CoA dehydratase, H) 2,3-dehydroadipyl-CoA hydrolase,transferase or ligase, I) muconate reductase. Abbreviations are:HODH=4-hydroxy-2-oxoheptane-1,7-dioate, OHED=2-oxohept-4-ene-1,7-dioate,6-OHE=6-oxo-2,3-dehydrohexanoate.

FIG. 5 shows pathways to muconate from lysine. Enzymes are A) lysineaminotransferase and/or aminating oxidoreductase, B) 2-aminoadipatesemialdehyde dehydrogenase, C) 2-aminoadipate deaminase, D) muconatereductase, E) lysine-2,3-aminomutase, F) 3,6-diaminohexanoateaminotransferase and/or aminating oxidoreductase, G) 3-aminoadipatesemialdehyde dehydrogenase, H) 3-aminoadipate deaminase.

FIG. 6 shows 3 thiolases with demonstrated thiolase activity resultingin acetoacetyl-CoA formation (left panel). FIG. 6 also shows thatseveral enzymes demonstrated selective condensation of succinyl-CoA andacetyl-CoA to form β-ketoadipyl-CoA as the sole product (right panel).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed, in part, to a biosynthetic pathwayfor synthesizing muconate from simple carbohydrate feedstocks, which inturn provides a viable synthetic route to PTA. In particular, pathwaysdisclosed herein provide trans,trans-muconate or cis,trans-muconatebiocatalytically from simple sugars. The all trans or cis,trans isomerof muconate is then converted to PTA in a two step process via inverseelectron demand Diels-Alder reaction with acetylene followed byoxidation in air or oxygen. The Diels-Alder reaction between muconateand acetylene proceeds to form cyclohexa-2,5-diene-1,4-dicarboxylate(P1), as shown in FIG. 1. Subsequent exposure to air or oxygen rapidlyconverts P1 to PTA.

This invention is also directed, in part, to non-naturally occurringmicroorganisms that express genes encoding enzymes that catalyzemuconate production. Pathways for the production of muconate disclosedherein are derived from central metabolic precursors. Successfullyengineering these pathways entails identifying an appropriate set ofenzymes with sufficient activity and specificity, cloning theircorresponding genes into a production host, optimizing the expression ofthese genes in the production host, optimizing fermentation conditions,and assaying for product formation following fermentation.

The maximum theoretical yield of muconic acid is 1.09 moles per moleglucose utilized. Achieving this yield involves assimilation of CO₂ asshown in equation 1 below:11C₆H₁₂O₆+6CO₂→12C₆H₆O₄+30H₂O  (equation 1)

As used herein, the term “muconate” is used interchangeably with muconicacid. Muconate is also used to refer to any of the possible isomericforms: trans,trans, cis,trans, and cis,cis. However, the presentinvention provides pathways to the useful trans,trans and cis,transforms, in particular.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a muconatebiosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having muconate biosyntheticcapability, those skilled in the art will understand with applying theteaching and guidance provided herein to a particular species that theidentification of metabolic modifications can include identification andinclusion or inactivation of orthologs. To the extent that paralogsand/or nonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a muconate pathway that includes at least oneexogenous nucleic acid encoding a muconate pathway enzyme expressed in asufficient amount to produce muconate. The muconate pathway includes anenzyme selected from the group consisting of a beta-ketothiolase, abeta-ketoadipyl-CoA hydrolase, a beta-ketoadipyl-CoA transferase, abeta-ketoadipyl-CoA ligase, a 2-fumarylacetate reductase, a2-fumarylacetate dehydrogenase, a trans-3-hydroxy-4-hexendioatedehydratase, a 2-fumarylacetate aminotransferase, a 2-fumarylacetateaminating oxidoreductase, a trans-3-amino-4-hexenoate deaminase, abeta-ketoadipate enol-lactone hydrolase, a muconolactone isomerase, amuconate cycloisomerase, a beta-ketoadipyl-CoA dehydrogenase, a3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA transferase, a2,3-dehydroadipyl-CoA hydrolase, a 2,3-dehydroadipyl-CoA ligase, amuconate reductase, a 2-maleylacetate reductase, a 2-maleylacetatedehydrogenase, a cis-3-hydroxy-4-hexendioate dehydratase, a2-maleylacetate aminotransferase, a 2-maleylacetate aminatingoxidoreductase, a cis-3-amino-4-hexendioate deaminase, and a muconatecis/trans isomerase.

In particular embodiments, the muconate pathway includes a set ofmuconate pathway enzymes shown in FIG. 2 and selected from the groupconsisting of:

A) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase, andbeta-ketoadipyl-CoA ligase, (3) beta-ketoadipate enol-lactone hydrolase,(4) muconolactone isomerase, (5) muconate cycloisomerase, and (6) amuconate cis/trans isomerase;

B) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase andbeta-ketoadipyl-CoA ligase, (3) 2-maleylacetate reductase, (4)2-maleylacetate dehydrogenase, (5) cis-3-hydroxy-4-hexendioatedehydratase, and (6) muconate cis/trans isomerase;

C) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase andbeta-ketoadipyl-CoA ligase, (3) 2-maleylacetate reductase, (4) an enzymeselected from 2-maleylacetate aminotransferase and 2-maleylacetateaminating oxidoreductase, (5) cis-3-amino-4-hexenoate deaminase, and (6)muconate cis/trans isomerase;

D) (1) beta-ketothiolase, (2) beta-ketoadipyl-CoA dehydrogenase, (3)3-hydroxyadipyl-CoA dehydratase, (4) an enzyme selected from2,3-dehydroadipyl-CoA transferase, 2,3-dehydroadipyl-CoA hydrolase and2,3-dehydroadipyl-CoA ligase, and (5) muconate reductase;

E) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase andbeta-ketoadipyl-CoA ligase, (3) 2-fumarylacetate reductase, (4)2-fumarylacetate dehydrogenase, and (5) trans-3-hydroxy-4-hexendioatedehydratase;

F) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase andbeta-ketoadipyl-CoA ligase, (3) 2-fumarylacetate reductase, (4) anenzyme selected from 2-fumarylacetate aminotransferase and2-fumarylacetate aminating oxidoreductase, and (5)trans-3-amino-4-hexenoate deaminase.

In some embodiments, a microbial organism having a pathway exemplifiedby those shown in FIG. 2 can include two or more exogenous nucleic acidseach encoding a muconate pathway enzyme, including three, four, five,six, that is up to all of the of enzymes in a muconate pathway. Thenon-naturally occurring microbial organism having at least one exogenousnucleic acid can include a heterologous nucleic acid. A non-naturallyoccurring microbial organism having a pathway exemplified by those shownin FIG. 2 can be cultured in a substantially anaerobic culture medium.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a muconate pathway that includes at least oneexogenous nucleic acid encoding a muconate pathway enzyme expressed in asufficient amount to produce muconate. The muconate pathway includes anenzyme selected from the group consisting of a 4-hydroxy-2-ketovaleratealdolase, a 2-oxopentenoate hydratase, a 4-oxalocrotonate dehydrogenase,a 2-hydroxy-4-hexenedioate dehydratase, a 4-hydroxy-2-oxohexanedioateoxidoreductase, a 2,4-dihydroxyadipate dehydratase (acting on2-hydroxy), a 2,4-dihydroxyadipate dehydratase (acting on 4-hydroxylgroup) and a 3-hydroxy-4-hexenedioate dehydratase.

In particular embodiments, the muconate pathway includes a set ofmuconate pathway enzymes shown in FIG. 3 and selected from the groupconsisting of:

A) (1) 4-hydroxy-2-ketovalerate aldolase, (2) 2-oxopentenoate hydratase,(3) 4-oxalocrotonate dehydrogenase, (4) 2-hydroxy-4-hexenedioatedehydratase;

B) (1) 4-hydroxy-2-ketovalerate aldolase, (2)4-hydroxy-2-oxohexanedioate oxidoreductase, (3) 2,4-dihydroxyadipatedehydratase (acting on 2-hydroxy), (4) 3-hydroxy-4-hexenedioatedehydratase; and

C) (1) 4-hydroxy-2-ketovalerate aldolase, (2)4-hydroxy-2-oxohexanedioate oxidoreductase, (3) 2,4-dihydroxyadipatedehydratase (acting on 4-hydroxyl group), (4) 2-hydroxy-4-hexenedioatedehydratase.

In some embodiments, a microbial organism having a pathway exemplifiedby those shown in FIG. 3 can include two or more exogenous nucleic acidseach encoding a muconate pathway enzyme, including three, four, that isup to all of the of enzymes in a muconate pathway. The non-naturallyoccurring microbial organism having at least one exogenous nucleic acidcan include a heterologous nucleic acid. A non-naturally occurringmicrobial organism having a pathway exemplified by those shown in FIG. 3can be cultured in a substantially anaerobic culture medium.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a muconate pathway that includes at least oneexogenous nucleic acid encoding a muconate pathway enzyme expressed in asufficient amount to produce muconate. The muconate pathway includes anenzyme selected from the group consisting of an HODH aldolase, an OHEDhydratase, an OHED decarboxylase, an HODH formate-lyase, an HODHdehydrogenase, an OHED formate-lyase, an OHED dehydrogenase, a 6-OHEdehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a2,3-dehydroadipyl-CoA hydrolase, a 2,3-dehydroadipyl-CoA transferase, a2,3-dehydroadipyl-CoA ligase, and a muconate reductase.

In particular embodiments, the muconate pathway includes a set ofmuconate pathway enzymes shown in FIG. 4 and selected from the groupconsisting of:

A) (1) HODH aldolase, (2) OHED hydratase, (3) OHED decarboxylase, (4)6-OHE dehydrogenase, and (5) muconate reductase;

B) (1) HODH aldolase, (2) OHED hydratase, (3) an enzyme selected fromOHED formate-lyase and OHED dehydrogenase, (4) an enzyme selected from2,3-dehydroadipyl-CoA hydrolase, 2,3-dehydroadipyl-CoA transferase and2,3-dehydroadipyl-CoA ligase, and (5) muconate reductase; and

C) (1) HODH aldolase, (2) an enzyme selected from HODH formate-lyase andHODH dehydrogenase, (3) 3-hydroxyadipyl-CoA dehydratase, (4) an enzymeselected from 2,3-dehydroadipyl-CoA hydrolase, 2,3-dehydroadipyl-CoAtransferase and 2,3-dehydroadipyl-CoA ligase, and (5) muconate reductase

In some embodiments, a microbial organism having a pathway exemplifiedby those shown in FIG. 4 can include two or more exogenous nucleic acidseach encoding a muconate pathway enzyme, including three, four, five,that is up to all of the of enzymes in a muconate pathway. Thenon-naturally occurring microbial organism having at least one exogenousnucleic acid can include a heterologous nucleic acid. A non-naturallyoccurring microbial organism having a pathway exemplified by those shownin FIG. 4 can be cultured in a substantially anaerobic culture medium.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having a muconate pathway that includes at least oneexogenous nucleic acid encoding a muconate pathway enzyme expressed in asufficient amount to produce muconate. The muconate pathway includes anenzyme selected from the group consisting of a lysine aminotransferase,a lysine aminating oxidoreductase, a 2-aminoadipate semialdehydedehydrogenase, a 2-aminoadipate deaminase, a muconate reductase, alysine-2,3-aminomutase, a 3,6-diaminohexanoate aminotransferase, a3,6-diaminohexanoate aminating oxidoreductase, a 3-aminoadipatesemialdehyde dehydrogenase, and a 3-aminoadipate deaminase.

In particular embodiments, the muconate pathway includes a set ofmuconate pathway enzymes shown in FIG. 5 and selected from the groupconsisting of:

A) (1) lysine aminotransferase, (2) lysine aminating oxidoreductase, (3)2-aminoadipate semialdehyde dehydrogenase, (4) 2-aminoadipate deaminase,and (5) muconate reductase

B) (1) lysine-2,3-aminomutase, (2) 3,6-diaminohexanoateaminotransferase, (3) 3,6-diaminohexanoate aminating oxidoreductase, (4)3-aminoadipate semialdehyde dehydrogenase, (5) 3-aminoadipate deaminase,and (6) muconate reductase.

In some embodiments, a microbial organism having a pathway exemplifiedby those shown in FIG. 5 can include two or more exogenous nucleic acidseach encoding a muconate pathway enzyme, including three, four, five,six, that is up to all of the of enzymes in a muconate pathway. Thenon-naturally occurring microbial organism having at least one exogenousnucleic acid can include a heterologous nucleic acid. A non-naturallyoccurring microbial organism having a pathway exemplified by those shownin FIG. 2 can be cultured in a substantially anaerobic culture medium.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a muconate pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting ofsuccinyl-CoA to beta-ketoadipyl-CoA, beta-ketoadipyl-CoA to3-hydroxyadipyl-CoA, 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA,2,3-dehydroadipyl-CoA to 2,3-dehydroadipate, and 2,3-dehydroadipate totrans,trans-muconate. Alternatively, the non-naturally occurringmicrobial organism comprises at least one exogenous nucleic acidencoding an enzyme or protein that converts a substrate to a productselected from the group consisting of succinyl-CoA tobeta-ketoadipyl-CoA, beta-ketoadipyl-CoA to beta-ketoadipate,beta-ketoadipate to 2-maleylacetate, 2-maleylacetate tocis-3-hydroxy-4-hexendioate, cis-3-hydroxy-4-hexendioate tocis,trans-muconate, and cis,trans-muconate to trans,trans-muconate.Alternatively, the non-naturally occurring microbial organism comprisesat least one exogenous nucleic acid encoding an enzyme or protein thatconverts a substrate to a product selected from the group consisting ofsuccinyl-CoA to beta-ketoadipyl-CoA, beta-ketoadipyl-CoA tobeta-ketoadipate, beta-ketoadipate to 2-maleylacetate, 2-maleylacetateto cis-3-amino-4-hexendioate, cis-3-amino-4-hexendioate tocis,trans-muconate, and cis,trans-muconate to trans,trans-muconate.Alternatively, the non-naturally occurring microbial organism comprisesat least one exogenous nucleic acid encoding an enzyme or protein thatconverts a substrate to a product selected from the group consisting ofsuccinyl-CoA to beta-ketoadipyl-CoA, beta-ketoadipyl-CoA tobeta-ketoadipate, beta-ketoadipate to 2-fumarylacetate, 2-fumarylacetateto trans-3-hydroxy-4-hexendioate, and trans-3-hydroxy-4-dienoate totrans,trans-muconate. Alternatively, the non-naturally occurringmicrobial organism comprises at least one exogenous nucleic acidencoding an enzyme or protein that converts a substrate to a productselected from the group consisting of succinyl-CoA tobeta-ketoadipyl-CoA, beta-ketoadipyl-CoA to beta-ketoadipate,beta-ketoadipate to 2-fumarylacetate, 2-fumarylacetate totrans-3-amino-4-hexendioate, trans-3-amino-4-hexendioate totrans,trans-muconate. Alternatively, the non-naturally occurringmicrobial organism comprises at least one exogenous nucleic acidencoding an enzyme or protein that converts a substrate to a productselected from the group consisting of succinyl-CoA tobeta-ketoadipyl-CoA, beta-ketoadipyl-CoA to beta-ketoadipate,beta-ketoadipate to beta-ketoadipate enol-lactone, beta-ketoadipateenol-lactone to muconolactone, muconolactone to cis,cis-muconate,cis,cis-muconate to cis,trans-muconate, and cis,trans muconate totrans,trans-muconate. Thus, the invention provides a non-naturallyoccurring microbial organism containing at least one exogenous nucleicacid encoding an enzyme or protein, where the enzyme or protein convertsthe substrates and products of a muconate pathway, such as those shownin FIG. 2.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a muconate pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of pyruvateand malonate semialdehyde to 4-hydroxy-2-oxohexandioate,4-hydroxy-2-oxohexandioate to 4-oxalocrotonate, 4-oxalocrotonate to2-hydroxy-4-hexendioate, and 2-hydroxy-4-hexendioate to muconate.Alternatively, the non-naturally occurring microbial organism comprisesat least one exogenous nucleic acid encoding an enzyme or protein thatconverts a substrate to a product selected from the group consisting ofpyruvate and malonate semialdehyde to 4-hydroxy-2-oxohexandioate,4-hydroxy-2-oxohexandioate, 4-hydroxy-2-oxohexandioate to2,4-dihydroxyadipate, 2,4-dihydroxyadipate to 2-hydroxy-4-hexendioate,and 2-hydroxy-4-hexendioate to muconate. Alternatively, thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of pyruvateand malonate semialdehyde to 4-hydroxy-2-oxohexandioate,4-hydroxy-2-oxohexandioate, 4-hydroxy-2-oxohexandioate to2,4-dihydroxyadipate, 2,4-dihydroxyadipate to 3-hydroxy-4-hexendioate,and 3-hydroxy-4-hexendioate to muconate. Thus, the invention provides anon-naturally occurring microbial organism containing at least oneexogenous nucleic acid encoding an enzyme or protein, where the enzymeor protein converts the substrates and products of a muconate pathway,such as those shown in FIG. 3.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a muconate pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of pyruvateand succinic semialdehyde to HODH, HODH to 3-hydroxyadipyl-CoA,3-hydroxy adipyl-CoA to 2,3-dehydroadipyl-CoA, 2,3-dehydroadipyl-CoA to2,3-dehydroadipate, and 2,3-dehydroadipate to muconate. Alternatively,the non-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of pyruvateand succinic semialdehyde to HODH, HODH to OHED, OHED to2,3-dehydroadipyl-CoA, 2,3-dehydroadipyl-CoA to 2,3-dehydroadipate, and2,3-dehydroadipate to muconate. Alternatively, the non-naturallyoccurring microbial organism comprises at least one exogenous nucleicacid encoding an enzyme or protein that converts a substrate to aproduct selected from the group consisting of pyruvate and succinicsemialdehyde to HODH, HODH to OHED, OHED to 6-OHE, 6-OHE to2,3-dehydroadipate, and 2,3-dehydroadipate to muconate. Thus, theinvention provides a non-naturally occurring microbial organismcontaining at least one exogenous nucleic acid encoding an enzyme orprotein, where the enzyme or protein converts the substrates andproducts of a muconate pathway, such as those shown in FIG. 4.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a muconate pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of lysine to2-aminoadipate semialdehyde, 2-aminoadipate semialdehyde to2-aminoadipate, 2-aminoadipate to 2,3-dehydroadipate, and2,3-dehydroadipate to muconate. Alternatively, the non-naturallyoccurring microbial organism comprises at least one exogenous nucleicacid encoding an enzyme or protein that converts a substrate to aproduct selected from the group consisting of lysine to3,6-diaminohexanoate, 3,6-diaminohexanoate to 3-aminoadipatesemialdehyde, 3-aminoadipate semialdehyde to 3-aminoadipate,3-aminoadipate to 2,3-dehydroadipate, and 2,3-dehydroadipate tomuconate. Thus, the invention provides a non-naturally occurringmicrobial organism containing at least one exogenous nucleic acidencoding an enzyme or protein, where the enzyme or protein converts thesubstrates and products of a muconate pathway, such as those shown inFIG. 5.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

Muconate can be produced from succinyl-CoA via beta-ketoadipate in aminimum of five enzymatic steps, shown in FIG. 2. In the first step ofall pathways, succinyl-CoA is joined to acetyl-CoA by abeta-ketothiolase to form beta-ketoadipyl-CoA (Step A). In oneembodiment, the beta-keto functional group is reduced and dehydrated toform 2,3-dehydroadipyl-CoA (Steps K and L). The CoA moiety is thenremoved by a CoA hydrolase, transferase or ligase to form2,3-dehydroadipate (Step M). Finally, 2,3-dehydroadipate is oxidized toform the conjugated diene muconate by an enoate oxidoreductase (Step N).

In other embodiments, beta-ketoadipyl-CoA is converted tobeta-ketoadipate by a CoA hydrolase, transferase or ligase (Step B).Beta-ketoadipate is then converted to 2-maleylacetate by maleylacetatereductase (Step O). The beta-ketone of 2-maleylacetate is then reducedto form cis-3-hydroxy-4-hexenoate (Step P). This product is furtherdehydrated to cis,trans-muconate in Step Q. Step W provides a muconatecis/trans-isomerase to provide trans,trans-muconate.

A similar route entails the conversion of 2-maleylacetate tocis-3-amino-4-hexenoate by an aminotransferase or aminatingoxidoreductase (Step R). Deamination of cis-3-amino-4-hexenoate issubsequently carried out to form cis,trans-muconate (Step S).

Alternatively, beta-ketoadipate can be converted to 2-fumarylacetate byaction of a fumarylacetate reductase (Step C). Such a reductase can beengineered by directed evolution, for example, of the correspondingmaleylacetate reductase. Reduction of the keto group and dehydrationprovides trans,trans-muconate (Steps D and E). Alternatively, reductiveamination, followed by deamination also affords the trans,trans-muconateproduct (Steps F and G)

In yet another route, beta-ketoadipate can be cyclized to anenol-lactone by beta-ketoadipyl enol-lactone hydrolase (Step H). Thedouble bond in the lactone ring is then shifted by muconolactoneisomerase (Step I). Finally, muconolactone is converted tocis,cis-muconate by muconate cycloisomerase (Step J). Muconatecycloisomerase may selectively form the cis,cis isomer of muconate.Further addition of a cis/trans isomerase converts the cis,cis isomer tothe favored trans, trans or trans, cis configurations (Steps T and W,which can be incorporated into a single isomerization step).

The pathways detailed in FIG. 2 can achieve a maximum theoretical yieldof 1.09 moles muconate per mole glucose utilized under anaerobic andaerobic conditions. With and without aeration, the maximum ATP yield is1 mole of ATP per glucose utilized at the maximum muconate yield. Thefirst step of this pathway, the condensation of succinyl-CoA andacetyl-CoA by beta-ketothiolase, has been demonstrated by Applicants isshown below in Example I.

Another pathway for muconate synthesis involves the condensation ofpyruvate and malonate semialdehyde, as shown in FIG. 3. Malonatesemialdehyde can be formed in the cell by several different pathways.Two example pathways are: 1) decarboxylation of oxaloacetate, and 2)conversion of 2-phosphoglycerate to glycerol which can then bedehydrated to malonate semialdehyde by a diol dehydratase. In onepathway, malonate semialdehyde and pyruvate are condensed to form4-hydroxy-2-oxohexanedioate (Step A). This product is dehydrated to form4-oxalocrotonate (Step B). 4-Oxalocrotonate is converted to muconate byreduction and dehydration of the 2-keto group (Steps C and D).

Alternately, the 2-keto group of 4-hydroxy-2-oxohexanedioate is reducedby an alcohol-forming oxidoreductase (Step E). The product,2,4-dihydroxyadipate is then dehydrated at the 2- or 4-hydroxy positionto form 2-hydroxy-4-hexenedioate (Step G) or 3-hydroxy-4-hexenedioate(Step F). Subsequent dehydration yields the diene, muconate (Steps D orH). This pathway is energetically favorable and is useful because itdoes not require carboxylation steps. Also, the pathway is driven by thestability of the muconate end product.

Several pathways for producing muconate from pyruvate and succinicsemialdehyde are detailed in FIG. 4. Such pathways entail aldolcondensation of pyruvate with succinic semialdehyde to4-hydroxy-2-oxoheptane-1,7-dioate (HODH) by HODH aldolase (Step A). Inone route, HODH is dehydrated to form 2-oxohept-4-ene-1,7-dioate (OHED)by OHED hydratase (Step B). OHED is then decarboxylated to form6-oxo-2,3-dehydrohexanoate (6-OHE) (Step C). This product issubsequently oxidized to the diacid and then further oxidized tomuconate (Steps F, I).

Alternately, HODH is converted to 3-hydroxyadipyl-CoA by a formate-lyaseor an acylating decarboxylating dehydrogenase (Step D). The 3-hydroxygroup of 3-hydroxyadipyl-CoA is then dehydrated to form the enoyl-CoA(Step G). The CoA moiety of 2,3-dehydroadipyl-CoA is removed by a CoAhydrolase, ligase or transferase (Step H). Finally, 2,3-dehydroadipateis oxidized to muconate by muconate reductase (Step I).

In yet another route, OHED is converted to 2,3-dehydroadipyl-CoA by aformate-lyase or acylating decarboxylating dehydrogenase (Step E).2,3-Dehydroadipyl-CoA is then transformed to muconate.

Pathways for producing muconate from lysine are detailed in FIG. 5. Inone embodiment, lysine is converted to 2-aminoadipate semialdehyde by anaminotransferase or aminating oxidoreductase (Step A). 2-Aminoadipatesemialdehyde is then oxidized to form 2-aminoadipate (Step B). The2-amino group is then deaminated by a 2-aminoadipate deaminase (Step C).The product, 2,3-dehydroadipate is further oxidized to muconate bymuconate reductase (Step D).

In an alternate route, the 2-amino group of lysine is shifted to the3-position by lysine-2,3-aminomutase (Step E). The product,3,6-diaminohexanoate, is converted to 3-aminoadipate semialdehyde by anaminotransferase or aminating oxidoreductase (Step F). Oxidation of thealdehyde (Step G) and deamination (Step H) yields 2,3-dehydroadipate,which is then converted to muconate (Step D).

All transformations depicted in FIGS. 2-5 fall into the generalcategories of transformations shown in Table 1. Below is described anumber of biochemically characterized genes in each category.Specifically listed are genes that can be applied to catalyze theappropriate transformations in FIGS. 2-5 when properly cloned andexpressed.

Table 1 shows the enzyme types useful to convert common centralmetabolic intermediates into muconate. The first three digits of eachlabel correspond to the first three Enzyme Commission number digitswhich denote the general type of transformation independent of substratespecificity.

TABLE 1 Label Function 1.1.1.a Oxidoreductase (oxo to alcohol, andreverse) 1.2.1.a Oxidoreductase (acid to oxo) 1.2.1.c Oxidoreductase(2-ketoacid to acyl-CoA) 1.3.1.a Oxidoreductase (alkene to alkane, andreverse) 1.4.1.a Oxidoreductase (aminating) 2.3.1.b Acyltransferase(beta-ketothiolase) 2.3.1.d Acyltransferase (formate C-acyltransferase)2.6.1.a Aminotransferase 2.8.3.a CoA transferase 3.1.1.a Enol-lactonehydrolase 3.1.2.a CoA hydrolase 4.1.1.a Carboxy-lyase 4.1.2.aAldehyde-lyase 4.2.1.a Hydro-lyase 4.3.1.a Ammonia-lyase 5.2.1.aCis/trans isomerase 5.3.3.a Lactone isomerase 5.4.3.a Aminomutase5.5.1.a Lactone cycloisomerase 6.2.1.a CoA synthetase

Several transformations depicted in FIGS. 2-5 require oxidoreductasesthat convert a ketone functionality to a hydroxyl group. The conversionof beta-ketoadipyl-CoA to 3-hydroxyadipyl-CoA (FIG. 2, Step K) iscatalyzed by a 3-oxoacyl-CoA dehydrogenase. The reduction of2-fumarylacetate to trans-3-hydroxy-4-hexendioate (FIG. 2, Step D) or2-maleylacetate to cis-3-hydroxy-4-hexendioate (FIG. 2, Step P), iscatalyzed by an oxidoreductase that converts a 3-oxoacid to a3-hydroxyacid. Reduction of the ketone group of 4-oxalocrotonate and4-hydroxy-2-oxohexanedioate to their corresponding hydroxyl group isalso catalyzed by enzymes in this family (FIG. 3, Steps C and E).

Exemplary enzymes for converting beta-ketoadipyl-CoA to3-hydroxyadipyl-CoA (FIG. 2, Step K) include 3-hydroxyacyl-CoAdehydrogenases. Such enzymes convert 3-oxoacyl-CoA molecules into3-hydroxyacyl-CoA molecules and are often involved in fatty acidbeta-oxidation or phenylacetate catabolism. For example, subunits of twofatty acid oxidation complexes in E. coli, encoded by fadB and fadJ,function as 3-hydroxyacyl-CoA dehydrogenases (Binstock and Schultz,Methods Enzymol. 71Pt C:403-411 (1981)). Furthermore, the gene productsencoded by phaC in Pseudomonas putida U (Olivera et al., Proc. Natl.Acad. Sci. U.S.A. 95:6419-6424 (1998)) and paaC in Pseudomonasfluorescens ST (Di et al., Arch. Microbiol. 188:117-125 (2007)) catalyzethe reverse reaction of step B in FIG. 10, that is, the oxidation of3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism ofphenylacetate or styrene. Note that the reactions catalyzed by suchenzymes are reversible. In addition, given the proximity in E. coli ofpaaH to other genes in the phenylacetate degradation operon (Nogales etal., Microbiology 153:357-365 (2007)) and the fact that paaH mutantscannot grow on phenylacetate (Ismail et al., Eur. J. Biochem.270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodesa 3-hydroxyacyl-CoA dehydrogenase. Genbank information related to thesegenes is summarized in Table 2 below.

TABLE 2 Gene GI # Accession No. Organism fadB 119811 P21177.2Escherichia coli fadJ 3334437 P77399.1 Escherichia coli paaH 16129356NP_415913.1 Escherichia coli phaC 26990000 NP_745425.1 Pseudomonasputida paaC 106636095 ABF82235.1 Pseudomonas fluorescens

Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoAmolecules to their corresponding 3-hydroxyacyl-CoA molecules include3-hydroxybutyryl-CoA dehydrogenases. The enzyme from Clostridiumacetobutylicum, encoded by hbd, has been cloned and functionallyexpressed in E. coli (Youngleson et al., J. Bacteriol. 171:6800-6807(1989)). Additional genes include Hbd1 (C-terminal domain) and Hbd2(N-terminal domain) in Clostridium kluyveri (Hillmer and Gottschalk,FEBS Lett. 21:351-354 (1972)) and HSD17B10 in Bos taurus (Wakil et al.,J. Biol. Chem. 207:631-638 (1954)). Yet other genes demonstrated toreduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloearamigera (Ploux et al., Eur. J. Biochem. 174:177-182 (1988)) and phaBfrom Rhodobacter sphaeroides (Alber et al., Mol. Microbiol. 61:297-309(2006)). The former gene is NADPH-dependent, its nucleotide sequence hasbeen determined (Peoples and Sinskey, Mol. Microbiol. 3:349-357 (1989))and the gene has been expressed in E. coli. Substrate specificitystudies on the gene led to the conclusion that it could accept3-oxopropionyl-CoA as an alternate substrate (Ploux et al., Eur. J.Biochem. 174:177-182 (1988)). Genbank information related to these genesis summarized in Table 3 below.

TABLE 3 Gene GI # Accession No. Organism hbd 18266893 P52041.2Clostridium acetobutylicum Hbd2 146348271 EDK34807.1 Clostridiumkluyveri Hbd1 146345976 EDK32512.1 Clostridium kluyveri HSD17B10 3183024O02691.3 Bos taurus phaB Rhodobacter sphaeroides phbB Zoogloea ramigera

A number of similar enzymes have been found in other species ofClostridia and in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)) as shown in Table 4.

TABLE 4 Gene GI # Accession No. Organism hbd 15895965 NP_349314.1Clostridium acetobutylicum hbd 20162442 AAM14586.1 Clostridiumbeijerinckii Msed_1423 146304189 YP_001191505 Metallosphaera sedulaMsed_0399 146303184 YP_001190500 Metallosphaera sedula Msed_0389146303174 YP_001190490 Metallosphaera sedula Msed_1993 146304741YP_001192057 Metallosphaera sedula

There are various alcohol dehydrogenases for converting 2-maleylacetateto cis-3-hydroxy-4-hexenoate (FIG. 2, Step P), 2-fumarylacetate totrans-3-hydroxy-4-hexenoate (FIG. 2, Step D), 4-oxalocrotonate to5-hydroxyhex-2-enedioate (FIG. 3, Step C) and4-hydroxy-2-oxohexanedioate to 2,4-dihydroxyadipate (FIG. 3, Step E).Two enzymes capable of converting an oxoacid to a hydroxyacid areencoded by the malate dehydrogenase (mdh) and lactate dehydrogenase(ldhA) genes in E. coli. In addition, lactate dehydrogenase fromRalstonia eutropha has been shown to demonstrate high activities onsubstrates of various chain lengths such as lactate, 2-oxobutyrate,2-oxopentanoate and 2-oxoglutarate (Steinbuchel and Schlegel, Eur. J.Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate intoalpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, anenzyme reported to be found in rat and in human placenta (Suda et al.,Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem.Biophys. Res. Commun. 77:586-591 (1977)). An additional gene for thesesteps is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) fromthe human heart which has been cloned and characterized (Marks et al.,J. Biol. Chem. 267:15459-15463 (1992)). This enzyme is a dehydrogenasethat operates on a 3-hydroxyacid. Another exemplary alcoholdehydrogenase converts acetone to isopropanol as was shown in C.beijerinckii (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003)) andT. brockii (Lamed and Zeikus, Biochem. J. 195:183-190 (1981); Peretz andBurstein, Biochemistry 28:6549-6555 (1989)). Genbank information relatedto these genes is summarized in Table 5 below.

TABLE 5 Gene GI # Accession No. Organism mdh 1789632 AAC76268.1Escherichia coli ldhA 16129341 NP_415898.1 Escherichia coli bdh 177198AAA58352.1 Homo sapiens adh 60592974 AAA23199.2 Clostridium beijerinckiiadh 113443 P14941.1 Thermoanaerobacter brockii

Enzymes in the 1.2.1 family are NAD(P)+-dependent oxidoreductases thatconvert aldehydes to acids. Reactions catalyzed by enzymes in thisfamily include the oxidation of 6-OHE (FIG. 4, Step F), 2-aminoadipatesemialdehyde (FIG. 5, Step B) and 3-aminoadipate semialdehyde (FIG. 5,Step G) to their corresponding acids. An exemplary enzyme is theNAD+-dependent aldehyde dehydrogenases (EC 1.2.1.3). Two aldehydedehydrogenases found in human liver, ALDH-1 and ALDH-2, have broadsubstrate ranges for a variety of aliphatic, aromatic and polycyclicaldehydes (Klyosov, A. A., Biochemistry 35:4457-4467 (1996)). ActiveALDH-2 has been efficiently expressed in E. coli using the GroELproteins as chaperonins (Lee et al., Biochem. Biophys. Res. Commun.298:216-224 (2002)). The rat mitochondrial aldehyde dehydrogenase alsohas a broad substrate range that includes the enoyl-aldehydecrotonaldehyde (Siew et al., Arch. Biochem. Biophys. 176:638-649(1976)). The E. coli gene astD also encodes an NAD+-dependent aldehydedehydrogenase active on succinic semialdehyde (Kuznetsova et al., FEMSMicrobiol. Rev. 29:263-279 (2005)). Genbank information related to thesegenes is summarized in Table 5 below.

TABLE 6 Gene GI # Accession No. Organism ALDH-2 118504 P05091.2 Homosapiens ALDH-2 14192933 NP_115792.1 Rattus norvegicus astD 3913108P76217.1 Escherichia coli

Two transformations in FIG. 4 require conversion of a 2-ketoacid to anacyl-CoA (FIG. 4, Steps D and E) by an enzyme in the EC class 1.2.1.Such reactions are catalyzed by multi-enzyme complexes that catalyze aseries of partial reactions which result in acylating oxidativedecarboxylation of 2-keto-acids. Exemplary enzymes that can be usedinclude 1) branched-chain 2-keto-acid dehydrogenase, 2)alpha-ketoglutarate dehydrogenase, and 3) the pyruvate dehydrogenasemultienzyme complex (PDHC). Each of the 2-keto-acid dehydrogenasecomplexes occupies positions in intermediary metabolism, and enzymeactivity is typically tightly regulated (Fries et al., Biochemistry42:6996-7002 (2003)). The enzymes share a complex but common structurecomposed of multiple copies of three catalytic components:alpha-ketoacid decarboxylase (E1), dihydrolipoamide acyltransferase (E2)and dihydrolipoamide dehydrogenase (E3). The E3 component is sharedamong all 2-keto-acid dehydrogenase complexes in an organism, while theE1 and E2 components are encoded by different genes. The enzymecomponents are present in numerous copies in the complex and utilizemultiple cofactors to catalyze a directed sequence of reactions viasubstrate channeling. The overall size of these dehydrogenase complexesis very large, with molecular masses between 4 and 10 million Da (i.e.,larger than a ribosome).

Activity of enzymes in the 2-keto-acid dehydrogenase family is normallylow or limited under anaerobic conditions in E. coli. Increasedproduction of NADH (or NADPH) could lead to a redox-imbalance, and NADHitself serves as an inhibitor to enzyme function. Engineering effortshave increased the anaerobic activity of the E. coli pyruvatedehydrogenase complex (Kim et al., Appl. Environ. Microbiol.73:1766-1771 (2001); Kim et al., J. Bacteriol. 190:3851-3858 (2008);Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). For example, theinhibitory effect of NADH can be overcome by engineering an H322Ymutation in the E3 component (Kim et al., J. Bacteriol. 190:3851-3858(2008)). Structural studies of individual components and how they worktogether in complex provide insight into the catalytic mechanisms andarchitecture of enzymes in this family (Aevarsson et al., Nat. Struct.Biol. 6:785-792 (1999); Zhou et al., Proc. Natl. Acad. Sci. U.S.A.98:14802-14807 (2001)). The substrate specificity of the dehydrogenasecomplexes varies in different organisms, but generally branched-chainketo-acid dehydrogenases have the broadest substrate range.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate tosuccinyl-CoA and is the primary site of control of metabolic fluxthrough the TCA cycle (Hansford, R. G., Curr. Top. Bioenerg. 10:217-278(1980)). Encoded by genes sucA, sucB and lpd in E. coli, AKGD geneexpression is downregulated under anaerobic conditions and during growthon glucose (Park et al., Mol. Microbiol. 15:473-482 (1993)). Althoughthe substrate range of AKGD is narrow, structural studies of thecatalytic core of the E2 component pinpoint specific residuesresponsible for substrate specificity (Knapp et al., J. Mol. Biol.280:655-668 (1998)). The Bacillus subtilis AKGD, encoded by odhAB (E1and E2) and pdhD (E3, shared domain), is regulated at thetranscriptional level and is dependent on the carbon source and growthphase of the organism (Resnekov et al., Mol. Gen. Genet. 234:285-296(1992)). In yeast, the LPD1 gene encoding the E3 component is regulatedat the transcriptional level by glucose (Roy and Dawes, J. Gen.Microbiol. 133:925-933 (1987)). The E1 component, encoded by KGD1, isalso regulated by glucose and activated by the products of HAP2 and HAP3(Repetto and Tzagoloff, Moll. Cell. Biol. 9:2695-2705 (1989)). The AKGDenzyme complex, inhibited by products NADH and succinyl-CoA, is known inmammalian systems, as impaired function of has been linked to severalneurological diseases (Tretter and dam-Vizi, Philos. Trans. R. Soc. LondB Biol. Sci. 360:2335-2345 (2005)). Genbank information related to thesegenes is summarized in Table 7 below.

TABLE 7 Gene GI # Accession No. Organism sucA 16128701 NP_415254.1Escherichia coli sucB 16128702 NP_415255.1 Escherichia coli lpd 16128109NP_414658.1 Escherichia coli odhA 51704265 P23129.2 Bacillus subtilisodhB 129041 P16263.1 Bacillus subtilis pdhD 118672 P21880.1 Bacillussubtilis KGD1 6322066 NP_012141.1 Saccharomyces cerevisiae KGD2 6320352NP_010432.1 Saccharomyces cerevisiae LPD1 14318501 NP_116635.1Saccharomyces cerevisiae

Branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as2-oxoisovalerate dehydrogenase, participates in branched-chain aminoacid degradation pathways, converting 2-keto acids derivatives ofvaline, leucine and isoleucine to their acyl-CoA derivatives and CO₂.The complex has been studied in many organisms including Bacillussubtilis (Wang et al., Eur. J. Biochem. 213:1091-1099 (1993)), Rattusnorvegicus (Namba et al., J. Biol. Chem. 244:4437-4447 (1969)) andPseudomonas putida (Sokatch et al., J. Bacteriol. 148:647-652 (1981)).In Bacillus subtilis the enzyme is encoded by genes pdhD (E3 component),bfmBB (E2 component), bfmBAA and bfmBAB (E1 component) (Wang et al.,Eur. J. Biochem. 213:1091-1099 (1993)). In mammals, the complex isregulated by phosphorylation by specific phosphatases and proteinkinases. The complex has been studied in rat hepatocites (Chicco et al.,J. Biol. Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha(E1 alpha), Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3components of the Pseudomonas putida BCKAD complex have beencrystallized (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999);Mattevi et al., Science 255:1544-1550 (1992)) and the enzyme complex hasbeen studied (Sokatch et al., J. Bacteriol. 148:647-652 (1981)).Transcription of the P. putida BCKAD genes is activated by the geneproduct of bkdR (Hesslinger et al., Mol. Microbiol. 27:477-492 (1998)).In some organisms including Rattus norvegicus (Paxton et al., Biochem.J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al.,Biochem. Mol. Biol. Int. 31:911-9122 (1993)), this complex has beenshown to have a broad substrate range that includes linear oxo-acidssuch as 2-oxobutanoate and alpha-ketoglutarate, in addition to thebranched-chain amino acid precursors. The active site of the bovineBCKAD was engineered to favor alternate substrate acetyl-CoA (Meng andChuang, Biochemistry 33:12879-12885 (1994)). Genbank information relatedto these genes is summarized in Table 8 below.

TABLE 8 Gene GI # Accession No. Organism bfmBB 16079459 NP_390283.1Bacillus subtilis bfmBAA 16079461 NP_390285.1 Bacillus subtilis bfmBAB16079460 NP_390284.1 Bacillus subtilis pdhD 118672 P21880.1 Bacillussubtilis lpdV 118677 P09063.1 Pseudomonas putida bkdB 129044 P09062.1Pseudomonas putida bkdA1 26991090 NP_746515.1 Pseudomonas putida bkdA226991091 NP_746516.1 Pseudomonas putida Bckdha 77736548 NP_036914.1Rattus norvegicus Bckdhb 158749538 NP_062140.1 Rattus norvegicus Dbt158749632 NP_445764.1 Rattus norvegicus Dld 40786469 NP_955417.1 Rattusnorvegicus

The pyruvate dehydrogenase complex, catalyzing the conversion ofpyruvate to acetyl-CoA, has also been studied. In the E. coli enzyme,specific residues in the E1 component are responsible for substratespecificity (Bisswanger, H., J. Biol. Chem. 256:815-822 (1981); Bremer,J., Eur. J. Biochem. 8:535-540 (1969); Gong et al., J. Biol. Chem.275:13645-13653 (2000)). As mentioned previously, enzyme engineeringefforts have improved the E. coli PDH enzyme activity under anaerobicconditions (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007);Kim et al., J. Bacteriol. 190:3851-3858 (2008); Zhou et al., Biotechnol.Letter. 30:335-342 (2008)). In contrast to the E. coli PDH, the B.subtilis complex is active and required for growth under anaerobicconditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). TheKlebsiella pneumoniae PDH, characterized during growth on glycerol, isalso active under anaerobic conditions (Menzel et al., J. Biotechnol.56:135-142 (1997)). Crystal structures of the enzyme complex from bovinekidney (Zhou et al., Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807(2001)) and the E2 catalytic domain from Azotobacter vinelandii areavailable (Mattevi et al., Science 255:1544-1550 (1992)). Some mammalianPDH enzymes complexes can react on alternate substrates such as2-oxobutanoate, although comparative kinetics of Rattus norvegicus PDHand BCKAD indicate that BCKAD has higher activity on 2-oxobutanoate as asubstrate (Paxton et al., Biochem. J. 234:295-303 (1986)). Genbankinformation related to these genes is summarized in Table 9 below.

TABLE 9 Gene GI # Accession No. Organism aceE 16128107 NP_414656.1Escherichia coli aceF 16128108 NP_414657.1 Escherichia coli lpd 16128109NP_414658.1 Escherichia coli pdhA 3123238 P21881.1 Bacillus subtilispdhB 129068 P21882.1 Bacillus subtilis pdhC 129054 P21883.2 Bacillussubtilis pdhD 118672 P21880.1 Bacillus subtilis aceE 152968699YP_001333808.1 Klebsiella pneumonia aceF 152968700 YP_001333809.1Klebsiella pneumonia lpdA 152968701 YP_001333810.1 Klebsiella pneumoniaPdha1 124430510 NP_001004072.2 Rattus norvegicus Pdha2 16758900NP_446446.1 Rattus norvegicus Dlat 78365255 NP_112287.1 Rattusnorvegicus Dld 40786469 NP_955417.1 Rattus norvegicus

As an alternative to the large multienzyme 2-keto-acid dehydrogenasecomplexes described above, some anaerobic organisms utilize enzymes inthe 2-ketoacid oxidoreductase family (OFOR) to catalyze acylatingoxidative decarboxylation of 2-keto-acids. Unlike the dehydrogenasecomplexes, these enzymes contain iron-sulfur clusters, utilize differentcofactors, and use ferredoxin or flavodoxin as electron acceptors inlieu of NAD(P)H. While most enzymes in this family are specific topyruvate as a substrate (POR) some 2-keto-acid:ferredoxinoxidoreductases have been shown to accept a broad range of 2-ketoacidsas substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukudaand Wakagi, Biochim. Biophys. Acta. 1597_74-80 (2002); Zhang et al., J.Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from thethermoacidophilic archaeon Sulfolobus tokodaii 7, which contains analpha and beta subunit encoded by gene ST2300 (Fukuda and Wakagi, supra;Zhang et al., supra). A plasmid-based expression system has beendeveloped for efficiently expressing this protein in E. coli (Fukuda etal., Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved insubstrate specificity were determined (Fukuda and Wakagi, supra). TwoOFORs from Aeropyrum pernix str. Kl have also been recently cloned intoE. coli, characterized, and found to react with a broad range of2-oxoacids (Nishizawa et al., FEBS Lett. 579_2319-2322 (2005)). The genesequences of these OFOR enzymes are available, although they do not haveGenBank identifiers assigned to date. There is bioinformatic evidencethat similar enzymes are present in all archaea, some anaerobic bacteriaand amitochondrial eukarya (Fukuda and Wakagi, supra). This class ofenzyme is also interesting from an energetic standpoint, as reducedferredoxin could be used to generate NADH by ferredoxin-NAD reductase(Petitdemange et al., Biochim. Biophys. Acta 421:334-337 (1976)). Also,since most of the enzymes are designed to operate under anaerobicconditions, less enzyme engineering may be required relative to enzymesin the 2-keto-acid dehydrogenase complex family for activity in ananaerobic environment. Genbank information related to these genes issummarized in Table 10 below.

TABLE 10 Gene GI # Accession No. Organism ST2300 15922633 NP_378302.1Sulfolobus tokodaii 7

Three transformations fall into the category of oxidoreductases thatreduce an alkene to an alkane (EC 1.3.1.-). The conversion ofbeta-ketoadipate to 2-maleylacetate (FIG. 2, Step O) is also catalyzedby the 2-enoate oxidoreductase maleylacetate reductase (MAR). A similarenzyme converts beta-ketoadipate to 2-fumarylacetate (FIG. 2, Step C).The oxidization of 2,3-dehydroadipate to muconate (FIG. 2, Step N) iscatalyzed by a 2-enoate oxidoreductase with muconate reductasefunctionality.

2-Enoate oxidoreductase enzymes are known to catalyze theNAD(P)H-dependent reduction and oxidation of a wide variety ofα,β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J. Biol.Chem. 276:5779-5787 (2001)). In the recently published genome sequenceof C. kluyveri, 9 coding sequences for enoate reductases were reported,out of which one has been characterized (Seedorf et al., Proc. Natl.Acad. Sci. U.S.A. 105:2128-2133 (2008)). The enr genes from both C.tyrobutyricum and M. thermoaceticum have been cloned and sequenced andshow 59% identity to each other. The former gene is also found to haveapproximately 75% similarity to the characterized gene in C. kluyveri(Giesel and Simon, Arch. Microbiol. 135:51-57 (1983)). It has beenreported based on these sequence results that enr is very similar to thedienoyl CoA reductase in E. coli (fadH) (Rohdich et al., J. Biol. Chem.276:5779-5787 (2001)). The C. thermoaceticum enr gene has also beenexpressed in a catalytically active form in E. coli (Rohdich et al.,supra). Genbank information related to these genes is summarized inTable 11 below.

TABLE 11 Gene GI # Accession No. Organism enr 169405742 ACA54153.1Clostridium botulinum A3 str enr 2765041 CAA71086.1 Clostridiumtyrobutyricum enr 3402834 CAA76083.1 Clostridium kluyveri enr 83590886YP_430895.1 Moorella thermoacetica fadH 16130976 NP_417552.1 Escherichiacoli

MAR is a 2-enoate oxidoreductase catalyzing the reversible reduction of2-maleylacetate (cis-4-oxohex-2-enedioate) to 3-oxoadipate (FIG. 2, StepO). MAR enzymes naturally participate in aromatic degradation pathways(Camara et al., J. Bacteriol. 191:4905-4915 (2009); Huang et al., Appl.Environ. Microbiol. 72:7238-7245 (2006); Kaschabek and Reineke, J.Bacteriol. 177:320-325 (1995); Kaschabek and Reineke, J. Bacteriol.175:6075-6081 (1993)). The enzyme activity was identified andcharacterized in Pseudomonas sp. strain B13 (Kaschabek and Reineke,(1995) supra; Kaschabek and Reineke, (1993) supra), and the coding genewas cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803(1997)). Additional MAR genes include clcE gene from Pseudomonas sp.strain B13 (Kasberg et al., supra), macA gene from Rhodococcus opacus(Seibert et al., J. Bacteriol. 175:6745-6754 (1993)), the macA gene fromRalstonia eutropha (also known as Cupriavidus necator) (Seibert et al.,Microbiology 150:463-472 (2004)), tfdFII from Ralstonia eutropha(Seibert et al., (1993) supra) and NCgl1112 in Corynebacteriumglutamicum (Huang et al., Appl. Environ Microbiol. 72:7238-7245 (2006)).A MAR in Pseudomonas reinekei MT1, encoded by ccaD, was recentlyidentified and the nucleotide sequence is available under the DBJ/EMBLGenBank accession number EF159980 (Camara et al., J. Bacteriol.191:4905-4915 (2009). Genbank information related to these genes issummarized in Table 12 below.

TABLE 12 Gene GI # Accession No. Organism clcE 3913241 O30847.1Pseudomonas sp. strain B13 macA 7387876 O84992.1 Rhodococcus opacus macA5916089 AAD55886 Cupriavidus necator tfdFII 1747424 AC44727.1 Ralstoniaeutropha JMP134 NCgl1112 19552383 NP_600385 Corynebacterium glutamicumccaD Pseudomonas reinekei MT1

In Step R of FIG. 2, 2-maleylacetate is transaminated to form3-amino-4-hexanoate. The conversion of 2-fumarylacetate totrans-3-amino-4-hexenedioate is a similar transformation (FIG. 2, StepF). These reactions are performed by aminating oxidoreductases in the ECclass 1.4.1. Enzymes in this EC class catalyze the oxidative deaminationof alpha-amino acids with NAD+ or NADP+ as acceptor, and the reactionsare typically reversible. Exemplary enzymes include glutamatedehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase(deaminating), encoded by ldh, and aspartate dehydrogenase(deaminating), encoded by nadX. The gdhA gene product from Escherichiacoli (Korber et al., J. Mol. Biol. 234:1270-1273 (1993); McPherson andWootton, Nucleic Acids Res. 11:5257-5266 (1983)), gdh from Thermotogamaritime (Kort et al., Extremophiles 1:52-60-1997); Lebbink et al., J.Mol. Biol. 280:287-296 (1998); Lebbink et al., J. Mol. Biol. 289:357-369(1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene349:237-244 (2005)) catalyze the reversible conversion of glutamate to2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both,respectively. The ldh gene of Bacillus cereus encodes the LeuDH proteinthat has a wide of range of substrates including leucine, isoleucine,valine, and 2-aminobutanoate (Ansorge and Kula, Biotechnol. Bioeng.68:557-562 (2000); Stoyan et al., J. Biotechnol. 54:77-80 (1997)). ThenadX gene from Thermotoga maritima encoding for the aspartatedehydrogenase is involved in the biosynthesis of NAD (Yang et al., J.Biol. Chem. 278:8804-8808 (2003)). Genbank information related to thesegenes is summarized in Table 13 below.

TABLE 13 Gene GI # Accession No. Organism gdhA 118547 P00370 Escherichiacoli gdh 6226595 P96110.4 Thermotoga maritima gdhA1 15789827 NP_279651.1Halobacterium salinarum ldh 61222614 P0A393 Bacillus cereus nadX15644391 NP_229443.1 Thermotoga maritima

The conversions of lysine to 2-aminoadipate semialdehyde (FIG. 5, StepA) and 3,6-diaminohexanoate to 3-aminoadipate semialdehyde (FIG. 5, StepF) are catalyzed by aminating oxidoreductases that transform primaryamines to their corresponding aldehydes. The lysine 6-dehydrogenase(deaminating), encoded by the lysDH genes, catalyze the oxidativedeamination of the 6-amino group of L-lysine to form2-aminoadipate-6-semialdehyde, which can spontaneously and reversiblycyclize to form Δ¹-piperideine-6-carboxylate (Misono and Nagasaki, J.Bacteriol. 150:398-401 (1982)). Exemplary enzymes are found inGeobacillus stearothermophilus (Heydari et al., Appl. Environ.Microbiol. 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto etal., J. Biochem. 106:76-80 (1989), Misono and Nagasaki, supra), andAchromobacter denitrificans (Ruldeekulthamrong et al., BMB. Rep.41:790-795 (2008)). Such enzymes can convert 3,6-diaminohexanoate to3-aminoadipate semialdehyde given the structural similarity between3,6-diaminohexanoate and lysine. Genbank information related to thesegenes is summarized in Table 14 below.

TABLE 14 Gene GI # Accession No. Organism lysDH 13429872 BAB39707Geobacillus stearothermophilus lysDH 15888285 NP_353966 Agrobacteriumtumefaciens lysDH 74026644 AAZ94428 Achromobacter denitrificans

FIG. 2, step A uses a 3-oxoadipyl-CoA thiolase, or equivalently,succinyl CoA:acetyl CoA acyl transferase (β-ketothiolase). The geneproducts encoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J.Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera etal., Proc. Natl. Acad. Sci. U.S.A. 95:6419-6424 (1998)), paaE inPseudomonas fluorescens ST (Di et al., Arch. Micbrobiol. 188:117-125(2007)), and paaJ from E. coli (Nogales et al., Microbiology 153:357-365(2007)) catalyze the conversion of 3-oxoadipyl-CoA into succinyl-CoA andacetyl-CoA during the degradation of aromatic compounds such asphenylacetate or styrene. Since beta-ketothiolase enzymes catalyzereversible transformations, these enzymes can also be employed for thesynthesis of 3-oxoadipyl-CoA. Several beta-ketothiolases were shown tohave significant and selective activities in the oxoadipyl-CoA formingdirection as shown in Example I below including bkt from Pseudomonasputida, pcaF and bkt from Pseudomonas aeruginosa PAO1, bkt fromBurkholderia ambifaria AMMD, paaJ from E. coli, and phaD from P. putida.Genbank information related to these genes is summarized in Table 15below.

TABLE 15 Gene GI # Accession No. Organism paaJ 16129358 NP_415915.1Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13)phaD 3253200 AAC24332.1 Pseudomonas putida pcaF 506695 AAA85138.1Pseudomonas putida paaE 106636097 ABF82237.1 Pseudomonas fluorescens bktBurkholderia ambifaria AMMD bkt Pseudomonas aeruginosa PAO1 pcaFPseudomonas aeruginosa PAO1

The acylation of ketoacids HODH and OHED to their corresponding CoAderivatives (FIG. 4, Steps D and E) and concurrent release of formate,is catalyzed by formate C-acyltransferase enzymes in the EC class 2.3.1.Enzymes in this class include pyruvate formate-lyase and ketoacidformate-lyase. Pyruvate formate-lyase (PFL, EC 2.3.1.54), encoded bypflB in E. coli, converts pyruvate into acetyl-CoA and formate. Theactive site of PFL contains a catalytically essential glycyl radicalthat is posttranslationally activated under anaerobic conditions byPFL-activating enzyme (PFL-AE, EC 1.97.1.4) encoded by pflA (Knappe etal., Proc. Natl. Acad. Sci. U.S.A. 81:1332-1335 (1984); Wong et al.,Biochemistry 32:14102-14110 (1993)). A pyruvate formate-lyase fromArchaeglubus fulgidus encoded by pflD has been cloned, expressed in E.coli and characterized (Lehtio and Goldman, Protein Eng Des Sel17:545-552 (2004)). The crystal structures of the A. fulgidus and E.coli enzymes have been resolved (Lehtio et al., J. Mol. Biol.357:221-235 (2006); Leppanen et al., Structure 7:733-744 (1999)).Additional PFL and PFL-AE enzymes are found in Clostridium pasteurianum(Weidner and Sawyers, J. Bacteriol. 178:2440-2444 (1996)) and theeukaryotic alga Chlamydomonas reinhardtii (Hemschemeier et al.,Eukaryot. Cell 7:518_526 (2008)). Keto-acid formate-lyase (EC 2.3.1.-),also known as 2-ketobutyrate formate-lyase (KFL) and pyruvateformate-lyase 4, is the gene product of tdcE in E. coli. This enzymecatalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formateduring anaerobic threonine degradation, and can also substitute forpyruvate formate-lyase in anaerobic catabolism (Simanshu et al., J.Biosci. 32:1195-1206 (2007)). The enzyme is oxygen-sensitive and, likePflB, requires post-translational modification by PFL-AE to activate aglycyl radical in the active site (Hesslinger et al., Mol. Microbiol.27:477-492 (1998)). Genbank information related to these genes issummarized in Table 16 below.

TABLE 16 Gene GI # Accession No. Organism pflB 16128870 NP_415423.1Escherichia coli pflA 16128869 NP_415422.1 Escherichia coli tdcE48994926 AAT48170.1 Escherichia coli pflD 11499044 NP_070278.1Archaeglubus fulgidus pfl 2500058 Q46266.1 Clostridium pasteurianum act1072362 CAA63749.1 Clostridium pasteurianum pfl1 159462978XP_001689719.1 Chlamydomonas reinhardtii pflA1 159485246 XP_001700657.1Chlamydomonas reinhardtii

Several reactions in FIGS. 2 and 5 are catalyzed by aminotransferases inthe EC class 2.6.1 (FIG. 2, Steps F and R and FIG. 5, Steps A and F).Such enzymes reversibly transfer amino groups from aminated donors toacceptors such as pyruvate and alpha-ketoglutarate. The conversion oflysine to 2-aminoadipate (FIG. 5, Step A) is naturally catalyzed bylysine-6-aminotransferase (EC 2.6.1.36). This enzyme function has beendemonstrated in yeast and bacteria. Enzymes from Candida utilis (Hammeret al J. Basic Microbiol. 32:21-27 (1992)), Flavobacterium lutescens(Fuji et al. J. Biochem. 128:391-397 (2000)) and Streptomycesclavuligenus (Romero et al. J. Ind. Microbiol. Biotechnol. 18:241-246(1997)) have been characterized. A recombinant lysine-6-aminotransferasefrom S. clavuligenus was functionally expressed in E. coli (Tobin et alJ. Bacteriol. 173:6223-6229 (1991)). The F. lutescens enzyme is specificto alpha-ketoglutarate as the amino acceptor (Soda et al. Biochemistry7:4110-4119 (1968)). Lysine-6-aminotransferase is also an enzyme thatcan catalyze the transamination of 3,6-diaminohexanoate (FIG. 5, StepF), as this substrate is structurally similar to lysine. Genbankinformation related to these genes is summarized in Table 17 below.

TABLE 17 Gene GI # Accession No. Organism lat 10336502 BAB13756.1Flavobacterium lutescens lat 153343 AAA26777.1 Streptomyces clavuligenus

In Steps R and F of FIG. 2 the beta-ketones of 2-maleylacetate and2-fumarylacetate, respectively, are converted to secondary amines.Beta-alanine/alpha-ketoglutarate aminotransferase (WO08027742) reactswith beta-alanine to form malonic semialdehyde, a 3-oxoacid similar instructure to 2-maleylacetate. The gene product of SkPYD4 inSaccharomyces kluyveri was shown to preferentially use beta-alanine asthe amino group donor (Andersen and Hansen, Gene 124:105-109 (1993)).SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABAaminotransferase, UGA1 (Ramos et al., Eur. J. Biochem. 149:401-404(1985)), whereas SkPYD4 encodes an enzyme involved in both -alanine andGABA transamination (Andersen and Hansen, supra).3-Amino-2-methylpropionate transaminase catalyzes the transformationfrom methylmalonate semialdehyde to 3-amino-2-methylpropionate. Theenzyme has been characterized in Rattus norvegicus and Sus scrofa and isencoded by Abat (Kakimoto et al., Biochim. Biophys. Acta 156:374-380(1968); Tamaki et al., Methods Enzymol. 324:376-389 (2000)). Genbankinformation related to these genes is summarized in Table 18 below.

TABLE 18 Gene GI # Accession No. Organism SkyPYD4 98626772 ABF58893.1Lachancea kluyveri SkUGA1 98626792 ABF58894.1 Lachancea kluyveri UGA16321456 NP_011533.1 Saccharomyces cerevisiae Abat 122065191 P50554.3Rattus norvegicus Abat 120968 P80147.2 Sus scrofa

Another enzyme that can catalyze the aminotransferase reactions in FIGS.2 and 5 is gamma-aminobutyrate transaminase (GABA transaminase), whichnaturally interconverts succinic semialdehyde and glutamate to4-aminobutyrate and alpha-ketoglutarate and is known to have a broadsubstrate range (Liu et al., Biochemistry 43:10896-10905 (2004);Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991); Schulz etal., Appl. Environ. Microbiol. 56:1-6 (1990)). E. coli has two GABAtransaminases, encoded by gabT (Bartsch and Schulz, J. Bacteriol.172:7035-7042 (1990)) and puuE (Kurihara et al., J. Biol. Chem.280:4602-4608 (2005)). GABA transaminases in Mus musculus, Pseudomonasfluorescens, and Sus scrofa have been shown to react with alternatesubstrates (Cooper, A. J., Methods Enzymol. 113:80-82 (1985); Scott andJakoby, J. Biol. Chem. 234:932-936 (19590. Genbank information relatedto these genes is summarized in Table 19 below.

TABLE 19 Gene GI # Accession No. Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 P_415818.1 Escherichia coli aba 37202121NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens aba 47523600 NP_999428.1 Sus scrofa

CoA transferases catalyze the reversible transfer of a CoA moiety fromone molecule to another. Conversion of beta-ketoadipyl-CoA tobeta-ketoadipate (FIG. 2, Step B) is accompanied by the acylation ofsuccinate by beta-ketoadipyl-CoA transferase. The de-acylation of2,3-dehydroadipyl-CoA (FIG. 2, Step M and FIG. 4, Step H) can also becatalyzed by an enzyme in the 2.8.3 family.

Beta-ketoadipyl-CoA transferase (EC 2.8.3.6), also known assuccinyl-CoA:3:oxoacid-CoA transferase, is encoded by pcaI and pcaJ inPseudomonas putida (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)).Similar enzymes based on homology exist in Acinetobacter sp. ADP1(Kowalchuk et al., Gene 146:23-30 (1994)). Additional exemplarysuccinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacterpylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997))and Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403(2007)). Genbank information related to these genes is summarized inTable 20 below.

TABLE 20 Gene GI # Accession No. Organism pcaI 24985644 AAN69545.1Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcaI50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1Acinetobacter sp. ADP1 pcaI 21224997 NP_630776.1 Streptomyces coelicolorpcaJ 21224996 NP_630775.1 Streptomyces coelicolor HPAG1_0676 108563101YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51 (1994)),substrates similar in structure to 2,3-dehydroadipyl-CoA. The genesencoding this enzyme are gctA and gctB. This enzyme has reduced butdetectable activity with other CoA derivatives including glutaryl-CoA,2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur.J. Biochem. 118:315-321 (1981)). The enzyme has been cloned andexpressed in E. coli (Mack, supra). Genbank information related to thesegenes is summarized in Table 21 below.

TABLE 21 Gene GI # Accession No. Organism gctA 559392 CAA57199.1Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcusfermentans

Other exemplary CoA transferases are catalyzed by the gene products ofcat1, cat2, and cat3 of Clostridium kluyveri which have been shown toexhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferaseactivity, respectively (Seedorf et al., Proc. Natl. Acad. Sci U.S.A.105:2128-2133 (2008); Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996)). Similar CoA transferase activities are also present inTrichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418(2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem.279:45337-45346 (2004)). Genbank information related to these genes issummarized in Table 22 below.

TABLE 22 Gene GI # Accession No. Organism cat1 729048 P38946.1Clostridium kluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3146349050 EDK35586.1 Clostridium kluyveri TVAG_395550 123975034XP_001330176 Trichomonas vaginalis G3 Tb11.02.0290 71754875 XP_828352Trypanosoma brucei

A CoA transferase that can utilize acetyl-CoA as the CoA donor isacetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit)and atoD (beta subunit) genes (Korolev et al., Acta Crystallagr. D.Biol. Crystallagr. 58:2116-2121 (2002); Vanderwinkel et al., Biochem.Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has a broadsubstrate range (Sramek and Frerman, Arch. Biochem. Biophys. 171:14-26(1975)) and has been shown to transfer the CoA moiety to acetate from avariety of branched and linear acyl-CoA substrates, includingisobutyrate (Matthies and Schink, Appl. Environ. Microbiol. 58:1435-1439(1992)), valerate (Vanderwinkel et al, supra) and butanoate(Vanderwinkel et al, supra). This enzyme is induced at thetranscriptional level by acetoacetate, so modification of regulatorycontrol may be necessary for engineering this enzyme into a pathway(Pauli and Overath, Eur. J. Biochem. 29:553-562 (1972)). Similar enzymesexist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl.Environ. Microbiol. 68:5186-5190 (2002)), Clostridium acetobutylicum(Cary et al., Appl. Environ. Microbiol. 56:1576-1583 (1990); Weisenbornet al., Appl. Environ. Microbiol. 55:323-329 (1989)), and Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol. Biochem.71:58-58 (2007)). Genbank information related to these genes issummarized in Table 23 below.

TABLE 23 Gene GI # Accession No. Organism atoA 2492994 P76459.1Escherichia coli atoD 2492990 P76458.1 Escherichia coli actA 62391407YP_226809.1 Corynebacterium glutamicum cg0592 62389399 YP_224801.1Corynebacterium glutamicum ctfA 15004866 NP_149326.1 Clostridiumacetobutylicum ctfB 15004867 NP_149327.1 Clostridium acetobutylicum ctfA31075384 AAP42564.1 Clostridium saccharoperbutylacetonicum ctfB 31075385AAP42565.1 Clostridium saccharoperbutylacetonicum

In Step H of FIG. 2, the lactonization of beta-ketoadipate to formβ-ketoadipate-enol-lactone is be catalyzed by the beta-ketoadipateenol-lactonase (EC-3.1.1.24). Beta-ketoadipate enol-lactonase alsoparticipates in the catechol branch of the beta-ketoadipate pathway todegrade aromatic compounds, in the reverse direction of that required inStep H of FIG. 2. This enzyme is encoded by the pcaD gene in Pseudomonasputida (Hughes et al., J. Gen Microbiol. 134:2877-2887 (1988)),Rhodococcus opacus (Eulberg et al., J. Bacteriol. 180:1072-1081 (1998))and Ralstonia eutropha. In Acinetobacter calcoaceticus, genes encodingtwo β-ketoadipate enol-lactone hydrolases were identified (Patel et al.,J. Biol. Chem. 250:6567 (1975)). Genbank information related to thesegenes is summarized in Table 24 below.

TABLE 24 Gene GI # Accession No. Organism ELH 6015088 Q59093Acinetobacter calcoaceticus ELH2 6166146 P00632 Acinetobactercalcoaceticus pcaD 24982842 AAN67003 Pseudomonas putida pcaD 75426718O67982 Rhodococcus opacus pcaD 75411823 javascript:Blast2 Ralstoniaeutropha (‘Q9EV41’)Q9EV45

The hydrolysis of acyl-CoA molecules to their corresponding acids iscarried out by acyl CoA hydrolase enzymes in the 3.1.2 family, alsocalled thioesterases. Several eukaryotic acetyl-CoA hydrolases (EC3.1.2.1) have broad substrate specificity and thus represent suitableenzymes for hydrolyzing beta-ketoadipyl-CoA and 2,3-dehydroadipyl-CoA(FIG. 2, Steps B and M and FIG. 4, Step H). For example, the enzyme fromRattus norvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun.71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA andmalonyl-CoA. The enzyme from the mitochondrion of the pea leaf also hasa broad substrate specificity, with demonstrated activity on acetyl-CoA,propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, andcrotonyl-CoA (Zeiher and Randall, Plant Physiol. 94:20-27 (1990)). Theacetyl-CoA hydrolase, ACH1, from S. cerevisiae represents anotherhydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)). Genbankinformation related to these genes is summarized in Table 25 below.

TABLE 25 Gene GI # Accession No. Organism acot12 18543355 NP_570103.1Rattus norvegicus ACH1 6319456 NP_009538 Saccharomyces cerevisiae

Another hydrolase is the human dicarboxylic acid thioesterase, acot8,which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA,sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem.280:38125-28132 (2005)) and the closest E. coli homolog, tesB, which canalso hydrolyze a broad range of CoA thioesters (Naggert et al., J. Biol.Chem. 266:11044-11050 (1991)). A similar enzyme has also beencharacterized in the rat liver (Deana, R., Biochem. Int. 26:767-773(1992)). Genbank information related to these genes is summarized inTable 26 below.

TABLE 26 Gene GI # Accession No. Organism tesB 16128437 NP_414986Escherichia coli acot8 3191970 CAA15502 Homo sapiens acot8 51036669NP_570112 Rattus norvegicus

Other potential E. coli thioester hydrolases include the gene productsof tesA (Bonner and Bloch, J. Biol. Chem. 247:3123-3133 (1972)), ybgC(Kuznetsova et al., FEBS Microbiol. Rev. 29:263-279 (2005); Zhuang etal., FEBS Lett. 516:161-163 (2002)), paaI (Song et al., J. Biol. Chem.281:11028-11038 (2006)), and ybdB (Leduc et al., J. Bacteriol.1889:7112-7126 (2007)). Genbank information related to these genes issummarized in Table 27 below.

TABLE 27 Gene GI # Accession No. Organism teas 16128478 NP_415027Escherichia coli ybgC 16128711 NP_415264 Escherichia coli paaI 16129357NP_415914 Escherichia coli ybdB 16128580 NP_415129 Escherichia coli

Yet another hydrolase is the glutaconate CoA-transferase fromAcidaminococcus fermentans. This enzyme was transformed by site-directedmutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA,acetyl-CoA and 3-butenoyl-CoA (Mack and Buckel, FEBS Lett. 405:209-212(1997)), compounds similar in structure to 2,3-dehydroadipyl-CoA. Thisindicates that the enzymes encoding succinyl-CoA:3-ketoacid-CoAtransferases and acetoacetyl-CoA:acetyl-CoA transferases can also serveas enzymes for this reaction step but would require certain mutations tochange their function. Genbank information related to these genes issummarized in Table 28 below.

TABLE 28 Gene GI # Accession No. Organism gctA 559392 CAA57199Acidaminococcus fermentans gctB 559393 CAA57200 Acidaminococcusfermentans

Step C of FIG. 4 is catalyzed by a 2-ketoacid decarboxylase thatgenerates 6-oxo-2,3-dehydrohexanoate (6-OHE) from2-oxohept-4-ene-1,7-dioate (OHED). The decarboxylation of keto-acids iscatalyzed by a variety of enzymes with varied substrate specificities,including pyruvate decarboxylase (EC 4.1.1.1), benzoylformatedecarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase andbranched-chain alpha-ketoacid decarboxylase. Pyruvate decarboxylase(PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholicfermentation, catalyzing the decarboxylation of pyruvate toacetaldehyde. The enzyme from Saccharomyces cerevisiae has a broadsubstrate range for aliphatic 2-keto acids including 2-ketobutyrate,2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (22). This enzymehas been extensively studied, engineered for altered activity, andfunctionally expressed in E. coli (Killenberg-Jabs et al., Eur. J.Biochem. 268:1698-1704 (2001); Li and Jordan, Biochemistry38:10004-10012 (1999); ter Schure et al., Appl. Environ. Microbiol64:1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc,also has a broad substrate range and has been a subject of directedengineering studies to alter the affinity for different substrates(Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The crystalstructure of this enzyme is available (Killenberg-Jabs, et al., supra).Other well-characterized PDC enzymes include the enzymes fromAcetobacter pasteurians (Chandra et al., Arch. Microbiol. 176:443-451(2001)) and Kluyveromyces lactis (Krieger et al., Eur. J. Biochem.269:3256-3263 (2002)). Genbank information related to these genes issummarized in Table 29 below.

TABLE 29 Gene GI # Accession No. Organism pdc 118391 P06672.1 Zymomonasmobilus pdc1 30923172 P06169 Saccharomyces cerevisiae pdc 20385191AM21208 Acetobacter pasteurians pdc1 52788279 Q12629 Kluyveromyceslactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broadsubstrate range and has been the target of enzyme engineering studies.The enzyme from Pseudomonas putida has been extensively studied andcrystal structures of this enzyme are available (Hasson et al.,Biochemistry 37:9918-9930 (1998); Polovnikova et al., Biochemistry42:1820-1830 (2003)). Site-directed mutagenesis of two residues in theactive site of the Pseudomonas putida enzyme altered the affinity (Km)of naturally and non-naturally occurring substrates (Siegert et al.,Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzymehave been further modified by directed engineering (Lingen et al.,Protein Eng. 15:585-593 (2002); Lingen et al., Chembiochem. 4:721-726(2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, hasalso been characterized experimentally (Barrowman et al., FEMSMicrobiology Letters 34:57-60 (1986)). Additional genes from Pseudomonasstutzeri, Pseudomonas fluorescens and other organisms can be inferred bysequence homology or identified using a growth selection systemdeveloped in Pseudomonas putida (Henning et al., Appl. Environ.Microbiol. 72:7510-7517 (2006)). Genbank information related to thesegenes is summarized in Table 30 below.

TABLE 30 Gene GI # Accession No. Organism mdlC 3915757 P20906.2Pseudomonas putida mdlC 81539678 Q9HUR2.1 Pseudomonas aeruginosa dpgB126202187 ABN80423.1 Pseudomonas stutzeri ilvB-1 70730840 YP_260581.1Pseudomonas fluorescens

A third enzyme capable of decarboxylating 2-oxoacids isalpha-ketoglutarate decarboxylase (KGD). The substrate range of thisclass of enzymes has not been studied to date. The KDC fromMycobacterium tuberculosis (Tian et al., Proc. Natl. Acad. Sci U.S.A.102:10670-10675 (2005)) has been cloned and functionally expressed,although it is large (˜130 kD) and GC-rich. KDC enzyme activity has beendetected in several species of rhizobia including Bradyrhizobiumjaponicum and Mesorhizobium loti (Green et al., J. Bacteriol.182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not beenisolated in these organisms, the genome sequences are available andseveral genes in each genome are annotated as putative KDCs. A KDC fromEuglena gracilis has also been characterized but the gene associatedwith this activity has not been identified to date (Shigeoka and Nakano,Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acidsstarting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV(Shigeoka and Nakano, supra). The gene could be identified by testinggenes containing this N-terminal sequence for KDC activity. Genbankinformation related to these genes is summarized in Table 31 below.

TABLE 31 Gene GI # Accession No. Organism kgd 160395583 O50463.4Mycobacterium tuberculosis kgd 27375563 NP_767092.1 Bradyrhizobiumjaponicum kgd 13473636 NP_105204.1 Mesorhizobium loti

A fourth enzyme for catalyzing this reaction is branched chainalpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shownto act on a variety of compounds varying in chain length from 3 to 6carbons (Oku and Kaneda, J. Bio. Chem. 263:18386-18396 (1988); Smit etal., App. Environ. Microbiol. 71:303-311 (2005)). The enzyme inLactococcus lactis has been characterized on a variety of branched andlinear substrates including 2-oxobutanoate, 2-oxohexanoate,2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate andisocaproate (Smit et al., supra). The enzyme has been structurallycharacterized (Berg et al., Science 318:1782-1786 (2007)). Sequencealignments between the Lactococcus lactis enzyme and the pyruvatedecarboxylase of Zymomonas mobilus indicate that the catalytic andsubstrate recognition residues are nearly identical (Siegert et al.,Protein Eng Des Sel 18:345-357 (2005)), so this enzyme can be subjectedto directed engineering. Decarboxylation of alpha-ketoglutarate by aBCKA was detected in Bacillus subtilis; however, this activity was low(5%) relative to activity on other branched-chain substrates (Oku andKaneda, supra) and the gene encoding this enzyme has not been identifiedto date. Additional BCKA genes can be identified by homology to theLactococcus lactis protein sequence. Many of the high-scoring BLASTphits to this enzyme are annotated as indolepyruvate decarboxylases (EC4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme thatcatalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde inplants and plant bacteria. Genbank information related to these genes issummarized in Table 32 below.

TABLE 32 Gene GI # Accession No. Organism kdcA 44921617 AAS49166.1Lactococcus lactis

Recombinant branched chain alpha-keto acid decarboxylase enzymes derivedfrom the E1 subunits of the mitochondrial branched-chain keto aciddehydrogenase complex from Homo sapiens and Bos taurus have been clonedand functionally expressed in E. coli (Davie et al., J. Biol. Chem.267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887(1992); Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). It wasindicated that co-expression of chaperonins GroEL and GroES enhanced thespecific activity of the decarboxylase by 500-fold (Wynn (1992) supra).These enzymes are composed of two alpha and two beta subunits. Genbankinformation related to these genes is summarized in Table 33 below.

TABLE 33 Gene GI # Accession No. Organism BCKDHB 34101272 NP_898871.1Homo sapiens BCKDHA 11386135 NP_000700.1 Homo sapiens BCKDHB 115502434P21839 Bos taurus BCKDHA 129030 P11178 Bos taurus

Aldehyde lyases in EC class 4.1.2 catalyze two key reactions in thedisclosed pathways to muconate (FIG. 3, Step A and FIG. 4, Step A). HOHDaldolase, also known as HHED aldolase, catalyzes the conversion of4-hydroxy-2-oxo-heptane-1,7-dioate (HOHD) into pyruvate and succinicsemialdehyde (FIG. 4, Step A). HODH aldolase is a divalent metalion-dependent class II aldolase, catalyzing the final step of4-hydroxyphenylacetic acid degradation in E. coli C, E. coli W, andother organisms. In the native context, the enzyme functions in thedegradative direction. The reverse (condensation) reaction isthermodynamically unfavorable; however the equilibrium can be shiftedthrough coupling HOHD aldolase with downstream pathway enzymes that workefficiently on reaction products. Such strategies have been effectivefor shifting the equilibrium of other aldolases in the condensationdirection (Nagata et al., Appl. Microbiol. Biotechnol. 44:432-438(1995); Pollard et al., App. Environ. Microbiol. 64:4093-4094 (1998)).The E. coli C enzyme, encoded by hpcH, has been extensively studied andhas recently been crystallized (Rea et al., J. Mol. Biol. 373:866-876(2007); Stringfellow et al., Gene 166:73-76 (1995)). The E. coli Wenzyme is encoded by hpaI (Prieto et al., J. Bacteriol. 178:111-120(1996)). Genbank information related to these genes is summarized inTable 34 below.

TABLE 34 Gene GI # Accession No. Organism hpcH 633197 CAA87759.1Escherichia coli C hpaI 38112625 AAR11360.1 Escherichia coli W

In Step A of FIG. 3, pyruvate and malonate semialdehyde are joined by analdehyde lyase to form 4-hydroxy-2-oxohexanedioate. An enzyme catalyzingthis exact reaction has not been characterized to date. A similarreaction is catalyzed by 2-dehydro-3-deoxyglucarate aldolase (DDGA, EC4.1.2.20), a type II aldolase that participates in the catabolic pathwayfor D-glucarate/galactarate utilization in E. coli. Tartronatesemialdehyde, the natural substrate of DDGA, is similar in size andstructure to malonate semialdehyde. This enzyme has a broad substratespecificity and has been shown to reversibly condense a wide range ofaldehydes with pyruvate (Fish and Blumenthal, Methods Enzymol. 9:529-534(1966)). The crystal structure of this enzyme has been determined and acatalytic mechanism indicated (Izard and Blackwell, EMBO J. 19:3849-3856(2000)). Other DDGA enzymes are found in Leptospira interrogans (Li etal., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun.62:1269-1270 (2006)) and Sulfolobus solfataricus (Buchanan et al.,Biochem. J. 343 Pt 3:563-570 (1999)). The S. solfataricus enzyme ishighly thermostable and was cloned and expressed in E. coli (Buchanan etal., supra). Genbank information related to these genes is summarized inTable 35 below.

TABLE 35 Gene GI # Accession No. Organism garL 1176153 P23522.2Escherichia coli LA_1624 24195249 AAN48823.1 Leptospira interrogansAJ224174.1:1..885 2879782 CAA11866.1 Sulfolobus solfataricus

The pathways in FIGS. 2-4 employ numerous enzymes in the dehydrataseclass of enzymes (EC 4.1.2). Several reactions in FIGS. 2 and 3 undergodehydration reactions similar to the dehydration of malate to fumarate,catalyzed by fumarate hydratase (EC 4.2.1.2). These transformationsinclude the dehydration of 3-hydroxy-4-hexenedioate (FIG. 2, Steps E andQ and FIG. 3, Step H), 4-hydroxy-2-oxohexanedioate (FIG. 3, Step B),2-hydroxy-4-hexenedioate (FIG. 3, Step D) and 2,4-dihydroxyadipate (FIG.3, Steps F and G). Fumarate hydratase enzymes are exemplary enzymes forcatalyzing these reactions. The E. coli fumarase encoded by fumCdehydrates a variety of alternate substrates including tartrate andthreo-hydroxyaspartate (Teipel et al., J. Biol. Chem. 243:5684-5694(1968)). A wealth of structural information is available for the E. colienzyme and researchers have successfully engineered the enzyme to alteractivity, inhibition and localization (Weaver, T., Acta Crystallogr. DBiol. Crystallagr. 61:1395-1401 (2005)). Exemplary fumarate hydrataseenzymes are found in Escherichia coli (Estevez et al., Protein Sci.11:1552-1557 (2002); Hong and Lee, Biotechnol. Bioprocess Eng. 9:252-255(2006); Rose and Weaver, Proc. Natl. Acad. Sci. U.S.A. 101:3393-3397(2004)); Agnihotri and Liu, Bioorg. Med. Chem. 11:9-20 (2003)),Corynebacterium glutamicum (Genda et al., Biosci. Biotechnol. Biochem.71:1102-1109 (2006)), Campylobacter jejuni (Smith and Gray, CatalysisLetters 6:195-199 (1990)), Thermus thermophilus (Mizobata et al., Arch.Biochem. Biophys. 355:49-55 (1998)), and Rattus norvegicus (Kobayashi etal., J. Biochem. 89:1923-1931 (1981)). Genbank information related tothese genes is summarized in Table 36 below.

TABLE 36 Gene GI # Accession No. Organism fumC 120601 P05042.1Escherichia coli K12 fumC 39931596 Q8NRN8.1 Corynebacterium glutamicumfumC 9789756 O69294.1 Campylobacter jejuni fumC 75427690 P84127 Thermusthennophilus fumH 120605 P14408.1 Rattus norvegicus

Another enzyme for catalyzing these reactions is citramalate hydrolyase(EC 4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate tomesaconate. This enzyme has been studied in Methanocaldococcusjannaschii in the context of the pyruvate pathway to 2-oxobutanoate,where it has been shown to have a broad substrate specificity (Drevlandet al., J. Bacteriol. 189:4391-4400 (2007)). This enzyme activity wasalso detected in Clostridium tetanomorphum, Morganella morganii,Citrobacter amalonaticus where it is thought to participate in glutamatedegradation (Kato and Asano Arch. Microbiol. 168:457-463 (1997)). The M.jannaschii protein sequence does not bear significant homology to genesin these organisms. Genbank information related to these genes issummarized in Table 37 below.

TABLE 37 Gene GI # Accession No. Organism leuD 3122345 Q58673.1Methanocaldococcus jannaschii

The enzyme OHED hydratase (FIG. 4, Step B) participates in4-hydroxyphenylacetic acid degradation, where it converts2-oxo-hept-4-ene-1,7-dioate (OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate(HODH) using magnesium as a cofactor (Burks et al., J. Am. Chem. Soc.120 (1998). OHED hydratase enzymes have been identified andcharacterized in E. coli C (Izumi et al., J. Mol. Biol. 370:899-911(2007); Roper et al., Gene 156:47-51 (1995)) and E. coli W (Prieto etal., J. Bacteriol. 178:111-120 (1996)). Sequence comparison revealshomologs in a range of bacteria, plants and animals. Enzymes with highlysimilar sequences are contained in Klebsiella pneumonia (91% identity,evalue=2e-138) and Salmonella enterica (91% identity, evalue=4e-138),among others. Genbank information related to these genes is summarizedin Table 38 below.

TABLE 38 Gene GI # Accession No. Organism hpcG   556840 CAA57202.1Escherichia coli C hpaH 757830 CAA86044.1 Escherichia coli W hpaH150958100 ABR80130.1 Klebsiella pneumoniae Sari_01896 160865156ABX21779.1 Salmonella enterica

Dehydration of 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA (FIG. 2,Step L and FIG. 4, Step G) is catalyzed by an enzyme with enoyl-CoAhydratase activity. 3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), alsocalled crotonase, is an enoyl-CoA hydratase that dehydrates3-hydroxyisobutyryl-CoA to form crotonyl-CoA (FIG. 3, step 2). Crotonaseenzymes are required for n-butanol formation in some organisms,particularly Clostridial species, and also comprise one step of the3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilic Archaeaof the genera Sulfolobus, Acidianus, and Metallosphaera. Exemplary genesencoding crotonase enzymes can be found in C. acetobutylicum (Atsumi etal., Metab. Eng. 10:305-211 (2008); Boynton et al., J. Bacteriol.178:3015-3024 (1996)), C. kluyveri (Hillmer and Gottschalk, FEBS Lett.21:351-354 (1972)), and Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)) though the sequence of the latter gene is notknown. Genbank information related to these genes is summarized in Table39 below.

TABLE 39 Gene GI # Accession No. Organism crt 15895969 NP_349318.1Clostridium acetobutylicum crt1 153953091 YP_001393856.1 Clostridiumkluyveri

Additional enoyl-CoA hydratases (EC 4.2.1.17) catalyze the dehydrationof a range of 3-hydroxyacyl-CoA substrates (Agnihotri and Liu, Bioorg.Med. Chem. 11:9-20 (2003); Conrad et al., J. Bacteriol. 118:103-111(1974); Roberts et al., Arch. Microbiol. 117:99-108 (1978)). Theenoyl-CoA hydratase of Pseudomonas putida, encoded by ech, catalyzes theconversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al.,supra). Additional enoyl-CoA hydratase enzymes are phaA and phaB, of P.putida, and paaA and paaB from P. fluorescens (Olivera et al., Proc.Natl. Acad. Sci U.S.A. 95:6419-6424 (1998)). The gene product of pimF inRhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratasethat participates in pimeloyl-CoA degradation (Harrison and Harwood,Microbiology 151:727-736 (2005)). Lastly, a number of Escherichia coligenes have been shown to demonstrate enoyl-CoA hydratase functionalityincluding maoC (Park and Lee, Appl. Biochem. Biotechnol. 113-116:335-346(2004)), paaF (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003);Park and Lee, supra; Park and Yup, Biotechnol. Bioeg 86:681-686 (2004))and paaG (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003); Park andLee, supra; Park and Yup, Biotechnol. Bioeg 86:681-686 (2004)). Genbankinformation related to these genes is summarized in Table 40 below.

TABLE 40 Gene GI # Accession No. Organism ech 26990073 NP_745498.1Pseudomonas putida paaA 26990002 NP_745427.1 Pseudomonas putida paaB26990001 NP_745426.1 Pseudomonas putida phaA 106636093 ABF82233.1Pseudomonas fluorescens phaB 106636094 ABF82234.1 Pseudomonasfluorescens pimF 39650635 CAE29158 Rhodopseudomonas palustris maoC16129348 NP_415905.1 Escherichia coli paaF 16129354 NP_415911.1Escherichia coli paaG 16129355 NP_415912.1 Escherichia coli

Alternatively, the E. coli gene products of fadA and fadB encode amultienzyme complex involved in fatty acid oxidation that exhibitsenoyl-CoA hydratase activity (Nakahigashi and Inokuchi, Nucleic AcidsRes. 18:4937 (1990); Yang, S. Y. J. Bacteriol. 173:7405-7406 (1991);Yang et al., Biochemistry 30:6788-6795 (1991)). Knocking out a negativeregulator encoded by fadR can be utilized to activate the fadB geneproduct (Sato et al., J. Biosci. Bioeng. 103:38-44 (2007)). The fadI andfadJ genes encode similar functions and are naturally expressed underanaerobic conditions (Campbell et al., Mol. Microbiol. 47:793-805(2003)). Genbank information related to these genes is summarized inTable 41 below.

TABLE 41 Gene GI # Accession No. Organism fadA 49176430 YP_026272.1Escherichia coli fadB 16131692 NP_418288.1 Escherichia coli fadI16130275 NP_416844.1 Escherichia coli fadJ 16130274 NP_416843.1Escherichia coli fadR 16129150 NP_415705.1 Escherichia coli

An enzyme in the ammonia-lyase family is required to deaminate3-amino-4-hexenedioate (FIG. 2, Steps G and S), 2-aminoadipate (FIG. 5,Step C) and 3-aminoadipate (FIG. 5, Step H). Enzymes catalyzing thisexact transformation has not been identified. However the threesubstrates bear structural similarity to aspartate, the native substrateof aspartase (EC 4.3.1.1.). Aspartase is a widespread enzyme inmicroorganisms, and has been characterized extensively (Wakil et al., J.Biol. Chem. 207:631-638 (1954)). The E. coli enzyme has been shown toreact with a variety of alternate substrates includingaspartatephenylmethylester, asparagine, benzyl-aspartate and malate (Maet al., Ann N.Y. Acad Sci. 672:60-65 (1992)). In addition, directedevolution was been employed on this enzyme to alter substratespecificity (Asano et al., Biomol. Eng. 22:95-101 (2005)). The crystalstructure of the E. coli aspartase, encoded by aspA, has been solved(Shi et al., Biochemistry 36:9136-9144 (1997)). Enzymes with aspartasefunctionality have also been characterized in Haemophilus influenzae(Sjostrom et al., Biochim. Biophys. Acta 1324:182-190 (1997)),Pseudomonas fluorescens (Takagi and Kisumi, J. Bacteriol. 161:1-6(1985)), Bacillus subtilis (Sjostrom et al., supra) and Serratiamarcescens (Takagi and Kisumi supra). Genbank information related tothese genes is summarized in Table 42 below.

TABLE 42 Gene GI # Accession No. Organism aspA 90111690 NP_418562Escherichia coli aspA 1168534 P44324.1 Haemophilus influenzae aspA114273 P07346.1 Pseudomonas fluorescens ansB 251757243 P26899.1 Bacillussubtilis aspA 416661 P33109.1 Serratia marcescens

Another deaminase enzyme is 3-methylaspartase (EC 4.3.1.2). This enzyme,also known as beta-methylaspartase and 3-methylaspartate ammonia-lyase,naturally catalyzes the deamination of threo-3-methylasparatate tomesaconate. The 3-methylaspartase from Clostridium tetanomorphum hasbeen cloned, functionally expressed in E. coli, and crystallized(Asuncion et al., Acta Crystallogr. D. Biol Crystallogr. 57:731-733(2001); Asuncion et al., J. Biol. Chem. 277:8306-8311 (2002); Botting etal. Biochemistry 27:2953-2955 (1988); Goda et al., Biochemistry31:10747-10756 (1992)). In Citrobacter amalonaticus, this enzyme isencoded by BAA28709 (Kato and Asano, Arch. Microbiol. 168:457-463(1997)). 3-methylaspartase has also been crystallized from E. coliYG1002 (Asano and Kato, FEBS Microbiol. Lett. 118:255-258 (1994))although the protein sequence is not listed in public databases such asGenBank. Sequence homology can be used to identify additional genes,including CTC_02563 in C. tetani and ECs0761 in Escherichia coliO157:H7. Genbank information related to these genes is summarized inTable 43 below.

TABLE 43 Gene GI # Accession No. Organism mal 259429 AAB24070.1Clostridium tetanomorphum BAA28709 3184397 BAA28709.1 Citrobacteramalonaticus CTC_02563 28212141 NP_783085.1 Clostridium tetani ECs076113360220 BAB34184.1 Escherichia coli O157:H7

In FIG. 2 Step J, muconolactone is converted to muconate by muconatecycloisomerase. However, muconate cycloisomerase usually results in theformation of cis,cis-muconate, which may be difficult for the subsequentDiels-Alder chemistry. The cis, trans- or trans, trans-isomers arepreferred. Therefore, the addition of a cis, trans isomerase may help toimprove the yield of terephthalic acid. Enzymes for similar isomericconversions include maleate cis,trans-isomerase (EC 5.2.1.1),maleylacetone cis-trans-isomerase (EC 5.2.1.2), and cis,trans-isomeraseof unsaturated fatty acids (Cti).

Maleate cis, trans-isomerase (EC 5.2.1.1) catalyzes the conversion ofmaleic acid in cis formation to fumarate in trans formation (Scher andJakoby, J. Biol. Chem. 244:1878-1882 (1969)). The Alcalidgenes faecalismaiA gene product has been cloned and characterized (Hatakeyeama et al.,Biochem. Biophys. Res. Commun. 239:74-79 (1997)). Other maleatecis,trans-isomerases are available in Serratia marcescens (Hatakeyama etal., Biosci. Biotechnol. Biochem. 64:1477-1485 (2000)), Ralstoniaeutropha and Geobacillus stearothermophilus. Genbank information relatedto these genes is summarized in Table 44 below.

TABLE 44 Gene GI # Accession No. Organism maiA 2575787 BAA23002Alcaligenes faecalis maiA 113866948 YP_725437 Ralstonia eutropha H16maiA 4760466 BAA77296 Geobacillus stearothermophilus maiA 8570038BAA96747.1 Serratia marcescens

Maleylacetone cis,trans-isomerase (EC 5.2.1.2) catalyzes the conversionof 4-maleyl-acetoacetate to 4-fumaryl-acetyacetate, a cis to transconversion. This enzyme is encoded by maiA in Pseudomonas aeruginosaFernandez-Canon and Penalva, J. Biol. Chem. 273:329-337 (1998)) andVibrio cholera (Seltzer, S., J. Biol. Chem. 248:215-222 (1973)). Asimilar enzyme was identified by sequence homology in E. coli O157.Genbank information related to these genes is summarized in Table 45below.

TABLE 45 Gene GI # Accession No. Organism maiA 15597203 NP_250697Pseudomonas aeruginosa maiA 15641359 NP_230991 Vibrio cholerae maiA189355347 EDU73766 Escherichia coli O157

The cti gene product catalyzes the conversion of cis-unsaturated fattyacids (UFA) to trans-UFA. The enzyme has been characterized in P. putida(Junker and Ramos, J. Bacteriol. 181:5693-5700 (1999)). Similar enzymesare found in Shewanella sp. MR-4 and Vibrio cholerae. Genbankinformation related to these genes is summarized in Table 46 below.

TABLE 46 Gene GI # Accession No. Organism cti 5257178 AAD41252Pseudomonas putida cti 113968844 YP_732637 Shewanella sp. MR-4 cti229506276 ZP_04395785 Vibrio cholerae

The endocyclic migration of the double bond in the structure ofβ-ketoadipate-enol-lactone to form muconolactone (FIG. 2, Step I) iscatalyzed by muconolactone isomerase (EC 5.3.3.4). Muconolactoneisomerase also participates in the catechol branch of the β-ketoadipatepathway to degrade aromatic compounds, at the reverse direction of StepG. Muconolactone isomerase is encoded by the catC gene. The Pseudomonasputida muconolactone isomerase was purified and partial amino acidsequences of cyanogen bromide fragments were determined (Meagher, R. B.,Biochim. Biophys. Acta 494:33-47 (1997)). A DNA fragment carrying thecatBCDE genes from Acinetobacter calcoaceticus was isolated bycomplementing P. putida mutants and the complemented activities wereexpressed constitutively in the recombinant P. putida strains (Shanleyet, al., J. Bacteriol. 165:557-563 (1986). The A. calcoaceticus catBCDEgenes were also expressed at high levels in Escherichia coli under thecontrol of a lac promoter (Shanley et al., supra). Theaniline-assimilating bacterium Rhodococcus sp. AN-22 CatC was purifiedto homogeneity and characterized as a homo-octamer with a molecular massof 100 kDa (Matsumura et al., Biochem. J. 393:219-226 (2006)). Thecrystal structure of P. putida muconolactone isomerase was solved(Kattie et al., J. Mol. Biol. 205:557-571 (1989)). Genbank informationrelated to these genes is summarized in Table 47 below.

TABLE 47 Gene GI # Accession No. Organism Q3LHT1 122612792 catCRhodococcus sp. AN-22 Q43932 5915883 catC Acinetobacter calcoaceticusQ9EV41 75464174 catC Ralstonia eutropha P00948 5921199 catC Pseudomonasputida Q9Z9Y5 75475019 catC Frateuria species ANA-18

Lysine 2,3-aminomutase (EC 5.4.3.2) converts lysine to(3S)-3,6-diaminohexanoate (FIG. 5, Step E), shifting an amine group fromthe 2- to the 3-position. The enzyme is found in bacteria that fermentlysine to acetate and butyrate, including as Fusobacterium nuleatum(kamA) (Barker et al., J. Bacteriol. 152:201-207 (1982)) and Clostridiumsubterminale (kamA) (Chirpich et al., J. Biol. Chem. 245:1778-1789(1970)). The enzyme from Clostridium subterminale has been crystallized(Lepore et al., Proc. Natl. Acad. Sci U.S.A. 102:13819-13824 (2005)). Anenzyme encoding this function is also encoded by yodO in Bacillussubtilus (Chen et al., Biochem. J. 348 Pt 3:539-549 (2000)). The enzymeutilizes pyridoxal 5′-phosphate as a cofactor, requires activation byS-Adenosylmethoionine, and is stereoselective, reacting with the onlywith L-lysine. Genbank information related to these genes is summarizedin Table 48 below.

TABLE 48 Gene GI # Accession No. Organism yodO 4033499 O34676.1 Bacillussubtilus kamA 75423266 Q9XBQ8.1 Clostridium subterminale kamA 81485301Q8RHX4 Fusobacterium nuleatum subsp. nuleatum

In Step H of FIG. 2, the ring opening reaction of muconolactone to formmuconate is catalyzed by muconate cycloisomerase (EC 5.5.1.1). Muconatecycloisomerase naturally converts cis,cis-muconate to muconolactone inthe catechol branch of the β-ketoadipate pathway to degrade aromaticcompounds. This enzyme has not been shown to react with the trans,transisomer. The muconate cycloisomerase reaction is reversible and isencoded by the catB gene. The Pseudomonas putida catB gene was clonedand sequenced (Aldrich et al., Gene 52:185-195 (1987)), the catB geneproduct was studied (Neidhart et al., Nature 347:692-694 (1990)) and itscrystal structures were resolved (Helin et al., J. Mol. Biol.254:918-941 (1995)). A DNA fragment carrying the catBCDE genes fromAcinetobacter calcoaceticus was isolated by complementing P. putidamutants and the complemented activities were expressed constitutively inthe recombinant P. putida strains (Shanley et al., J. Bacteriol.165:557-563 (1986)). The A. calcoaceticus catBCDE genes were alsoexpressed at high levels in Escherichia coli under the control of a lacpromoter (Shanley et al., supra). The Rhodococcus sp. AN-22 CatB waspurified to homogeneity and characterized as a monomer with a molecularmass of 44 kDa. The enzyme was activated by Mn²⁺, Co²⁺ and Mg²⁺(Matsumura et al., Biochem. J. 393:219-226 (2006)). Muconatecycloisomerases from other species, such as Rhodococcus rhodochrous N75,Frateuria species ANA-18, and Trichosporon cutaneum were also purifiedand studied (Cha and Bruce, FEMS Microbiol. Lett. 224:29-34 2003); Mazuret al., Biochemistry 33:1961-1970 (1994); Murakami et al., BiosciBiotechnol. Biochem. 62:1129-1133 (1998)). Genbank information relatedto these genes is summarized in Table 49 below.

TABLE 49 Gene GI # Accession No. Organism catB P08310 115713 Pseudomonasputida catB Q43931 51704317 Acinetobacter calcoaceticus catB Q3LHT2122612793 Rhodococcus sp. AN-22 catB Q9Z9Y1 75424020 Frateuria speciesANA-18 catB P46057 1170967 Trichosporon cutaneum

The conversion of beta-ketoadipyl-CoA to beta-ketoadipate (FIG. 2, StepB) and 2,3-dehydroadipyl-CoA to 2,3-dehydroadipate (FIG. 2, Step M andFIG. 4, Step H) can be catalyzed by a CoA acid-thiol ligase or CoAsynthetase in the 6.2.1 family of enzymes. Enzymes catalyzing theseexact transformations have not been characterized to date; however,several enzymes with broad substrate specificities have been describedin the literature. ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13)is an enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concomitant synthesis of ATP. ACD I fromArchaeoglobus fulgidus, encoded by AF1211, was shown to operate on avariety of linear and branched-chain substrates including isobutyrate,isopentanoate, and fumarate (Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)). A second reversible ACD in Archaeoglobus fulgidus,encoded by AF1983, was also shown to have a broad substrate range withhigh activity on cyclic compounds phenylacetate and indoleacetate(Musfeldt and Shonheit, supra). The enzyme from Haloarcula marismortui(annotated as a succinyl-CoA synthetase) accepts propionate, butyrate,and branched-chain acids (isovalerate and isobutyrate) as substrates,and was shown to operate in the forward and reverse directions (Brasenand Schonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded byPAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilumshowed the broadest substrate range of all characterized ACDs, reactingwith acetyl-CoA, isobutyryl-CoA (preferred substrate) andphenylacetyl-CoA (Brasen and Schonheit, supra). Directed evolution orengineering can be used to modify this enzyme to operate at thephysiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned,functionally expressed, and characterized in E. coli (Brasen andShonheit, supra; Musfeldt and Schonheit, supra). An additional enzyme isencoded by sucCD in E. coli, which naturally catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Buck et al., Biochemistry24:6245-6252 (1985)). Genbank information related to these genes issummarized in Table 50 below.

TABLE 50 Gene GI # Accession No. Organism AF1211 11498810 NP_070039.1Archaeoglobus fulgidus DSM 4304 AF1983 11499565 NP_070807.1Archaeoglobus fulgidus DSM 4304 scs 55377722 YP_135572.1 Haloarculamarismortui PAE3250 18313937 NP_560604.1 Pyrobaculum aerophilum str. IM2sucC 16128703 NP_415256.1 Escherichia coli sucD 1786949 AAC73823.1Escherichia coli

Another enzyme for this step is 6-carboxyhexanoate-CoA ligase, alsoknown as pimeloyl-CoA ligase (EC 6.2.1.14), which naturally activatespimelate to pimeloyl-CoA during biotin biosynthesis in gram-positivebacteria. The enzyme from Pseudomonas mendocina, cloned into E. coli,was shown to accept the alternate substrates hexanedioate andnonanedioate (Binieda et al, Biochem. J. 340 Pt 3:793-801 (1999)). Otherenzymes are found in Bacillus subtilis (Bower et al., J. Bacteriol.178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerly Bacillussphaericus) (Ploux et al., Biochem. J. 287 Pt 3:685-690 (1992)). Genbankinformation related to these genes is summarized in Table 51 below.

TABLE 51 Gene GI # Accession No. Organism pauA 15596214 NP_249708.1Pseudomonas mendocina bioW 50812281 NP_390902.2 Bacillus subtilis bioW115012 P22822.1 Lysinibacillus sphaericus

Additional CoA-ligases include the rat dicarboxylate-CoA ligase forwhich the sequence is yet uncharacterized (Vamecq et al., Biochem. J.230:683-693 (1985)), either of the two characterized phenylacetate-CoAligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J.395:147-155 (2006); Wang et al., Biochem. Biophys. Res. Commun.360:453-458 (2007)) and the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)).Acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim.Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al.,Biochem. Pharmacol. 65:989-994 (2003)) naturally catalyze theATP-dependant conversion of acetoacetate into acetoacetyl-CoA. Genbankinformation related to these genes is summarized in Table 52 below.

TABLE 52 Gene GI # Accession No. Organism phl 77019264 CAJ15517.1Penicillium chrysogenum phlB 152002983 ABS19624.1 Penicilliumchrysogenum paaF 22711873 AAC24333.2 Pseudomonas putida AACS 21313520NP_084486.1 Mus musculus AACS 31982927 NP_076417.2 Homo sapiens

In some embodiments, the present invention provides a semi-syntheticmethod for synthesizing terephthalate (PTA) that includes preparingmuconic acid by culturing the above-described organisms, reacting theresultant muconic acid with acetylene to form a cyclohexadiene adduct(P1, FIG. 1), and oxidizing the cyclohexadiene adduct to form PTA.Semi-synthetic methods combine the biosynthetic preparation of advancedintermediates with conventional organic chemical reactions.

While the culturing of muconic acid is discussed further below, theDiels-Alder reaction conditions are detailed here. Diels-Alder reactionsare widespread in the chemical industry and are known to those skilledin the art (Carruthers, W., Some Modern Methods of Organic Synthesis,Cambridge University Press (1986); Norton, J., Chem. Review 31:319-523(1942); Sauer, J., Angewandte Chemie 6:16-33 (1967)). This class ofpericyclic reactions is well-studied for its ability to generate cycliccompounds at low energetic cost. Diels-Alder reactions are thus anattractive and low-cost way of making a variety of pharmaceuticals andnatural products.

In a Diels-Alder reaction, a conjugated diene or heterodiene reacts withan alkene, alkyne, or other unsaturated functional group, known as adienophile, to form a six-membered ring. One aspect of the Diels-Alderreaction is that the two components usually have complementaryelectronic character, as determined by the energies of the highestoccupied molecular orbital (HOMO) and lowest unoccupied molecularorbital (LUMO) of the diene and dienophile (Carruthers, W., Some ModernMethods of Organic Synthesis, Cambridge University Press (1986). Innormal mode, the diene is electron-rich and the dienophile iselectron-poor, although this is not always the case. The method of thepresent invention provides the opposite electronic configuration with anelectron poor diene and a relatively electron rich dienophile, in whatis termed an inverse electron demand Diels-Alder reaction. The mainphysical constraint for this type of reaction is that the conjugateddiene must be able to adopt a cisoid conformation for the reaction toproceed. A wide variety of substituted conjugated dienes and dienophilesare able to undergo this chemistry.

In the disclosed reaction of FIG. 1, muconate is the conjugated diene,and is beneficially in the trans,trans or cis,trans isomericconfiguration for the reaction to proceed. The cis, cis isomer ofmuconate, prevalent in biological systems as a degradation product ofcatechol, is unlikely to adopt the required cisoid conformation due tosteric hindrance of the carboxylic acid groups. The trans,trans isomerof muconate (shown in FIG. 1) is able to react in Diels-Alder reactionswith a variety of dienes (Deno, N. C., J. Am. Chem. Soc. 72:4057-4059(1950); Sauer, J., Angewandte Chemie 6:16-33 (1967)).

Acetylene serves as the dienophile in the production of PTA. Acetyleneand substituted acetylene derivatives are well-known dienophiles((Carruthers, W., Some Modern Methods of Organic Synthesis, CambridgeUniversity Press (1986); U.S. Pat. No. 3,513,209, Clement, R. A.; Dai etal., J. Am. Chem. Soc. 129:645-657 (2007)). The addition ofelectron-withdrawing substituents increases reactivity in normal modeDiels Alder reactions; likewise, in the inverse electron demand,electron donating groups are employed to increase reactivity. Atelevated temperatures, unsubstituted acetylene has been shown to reactwith butadiene and other substituted linear and cyclic dienes (U.S. Pat.No. 3,513,209, Clement, R. A.; Norton, J., Chem. Review 31:319-523(1942); Vijaya et al., J. Mol. Struct. 589-590:291-299 (2002)).

Increased temperature can be used to perform the Diels-Alder reaction inFIG. 1. For example, the Diels-Alder reaction of acetylene with1,3-butadiene to form 1,4-cyclohexadiene is performed in the range of80-300° C. (U.S. Pat. No. 3,513,209, Clement, R. A. supra).

Other reaction conditions that have been shown to enhance the rate ofDiels-Alder reactions include elevated pressure, the addition of a Lewisacid, and stoichiometric excess of acetylene. Elevated pressure up to1000 atmospheres was shown to enhance the rate of 1,4-cyclohexadieneformation from butadiene and acetylene (U.S. Pat. No. 3,513,209,Clement, R. A.). Catalytic amounts of Lewis acids can also improvereaction rate (Nicolaou et al., Angewandte Chemie 41:1668-1698 (2002)).Some suitable Lewis acids include magnesium halides such as magnesiumchloride, magnesium bromide or magnesium iodide or zinc halides such aszinc chloride, zinc bromide or zinc iodide. Stoichiometric excess ofacetylene will aid in reducing formation of homopolymerizationbyproducts.

Oxidation of the Diels-Alder product,cyclohexa-2,5-diene-1,4-dicarboxylate (P1), to PTA can be accomplishedin the presence or absence of catalyst under mild reaction conditions.The driving force for P1 oxidation is the formation of the aromatic ringof PTA. Precedence for the conversion of P1 to PTA in the absence ofcatalyst is the conversion of 1,4-cyclohexadiene to benzene in air (U.S.Pat. No. 3,513,209, Clement, R. A.). 1,4-Cyclohexadiene is alsoconverted to benzene by catalysis, for example using transition metalcomplexes such as bis(arene)molybdenum(0) and bis(arene)chromium(0)(Fochi, G., Organometallics 7:225-2256 (1988)) or electroactivebinuclear rhodium complexes (Smith and Gray, Catalysis Letters 6:195-199(1990)).

In some embodiments, the method for synthesizing PTA includes isolatingmuconic acid from the culture broth prior to reacting with acetylene inthe Diels-Alder reaction. This is particularly helpful since theDiels-Alder reaction, is frequently done in the absence of a solvent,especially under thermal conditions. Isolation of muconic acid caninvolve various filtration and centrifugation techniques. Cells of theculture and other insoluble materials can be filtered viaultrafiltration and certain salts can be removed by nanofiltration.Because muconic acid is a diacid, standard extraction techniques can beemployed that involve adjusting the pH. After removal of substantiallyall solids and salts, the muconic acid can be separated from water byremoval of water with heating in vacuo, or by extraction at low pH. Forexample, following the addition of sulfuric acid or phosphoric acid tothe fermentation broth in sufficient amounts (pH 3 or lower), the freecarboxylate acid form of muconic acid precipitates out of solution (U.S.Pat. No. 4,608,338). In this form, muconic acid is readily separatedfrom the aqueous solution by filtration or other conventional means.

In some embodiments, the muconic acid need not be isolated. Instead, theDiels-Alder reaction between muconic acid and acetylene can be performedin the culture broth. In such a case, the culture broth can beoptionally filtered prior to adding acetylene.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more muconatebiosynthetic pathways. Depending on the host microbial organism chosenfor biosynthesis, nucleic acids for some or all of a particular muconatebiosynthetic pathway can be expressed. For example, if a chosen host isdeficient in one or more enzymes or proteins for a desired biosyntheticpathway, then expressible nucleic acids for the deficient enzyme(s) orprotein(s) are introduced into the host for subsequent exogenousexpression. Alternatively, if the chosen host exhibits endogenousexpression of some pathway genes, but is deficient in others, then anencoding nucleic acid is needed for the deficient enzyme(s) orprotein(s) to achieve muconate biosynthesis. Thus, a non-naturallyoccurring microbial organism of the invention can be produced byintroducing exogenous enzyme or protein activities to obtain a desiredbiosynthetic pathway or a desired biosynthetic pathway can be obtainedby introducing one or more exogenous enzyme or protein activities that,together with one or more endogenous enzymes or proteins, produces adesired product such as muconate.

Depending on the muconate biosynthetic pathway constituents of aselected host microbial organism, the non-naturally occurring microbialorganisms of the invention will include at least one exogenouslyexpressed muconate pathway-encoding nucleic acid and up to all encodingnucleic acids for one or more muconate biosynthetic pathways. Forexample, muconate biosynthesis can be established in a host deficient ina pathway enzyme or protein through exogenous expression of thecorresponding encoding nucleic acid. In a host deficient in all enzymesor proteins of a muconate pathway, exogenous expression of all enzyme orproteins in the pathway can be included, although it is understood thatall enzymes or proteins of a pathway can be expressed even if the hostcontains at least one of the pathway enzymes or proteins. For example,exogenous expression of all enzymes or proteins in a pathway forproduction of muconate can be included, such as those shown in FIGS.2-5.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the muconatepathway deficiencies of the selected host microbial organism. Therefore,a non-naturally occurring microbial organism of the invention can haveone, two, three, four, six, etc. up to all nucleic acids encoding theenzymes or proteins constituting a muconate biosynthetic pathwaydisclosed herein. In some embodiments, the non-naturally occurringmicrobial organisms also can include other genetic modifications thatfacilitate or optimize muconate biosynthesis or that confer other usefulfunctions onto the host microbial organism. One such other functionalitycan include, for example, augmentation of the synthesis of one or moreof the muconate pathway precursors such as succinyl-CoA.

Generally, a host microbial organism is selected such that it producesthe precursor of a muconate pathway, either as a naturally producedmolecule or as an engineered product that either provides de novoproduction of a desired precursor or increased production of a precursornaturally produced by the host microbial organism. For example,succinyl-CoA is produced naturally in a host organism such as E. coli. Ahost organism can be engineered to increase production of a precursor,as disclosed herein. In addition, a microbial organism that has beenengineered to produce a desired precursor can be used as a host organismand further engineered to express enzymes or proteins of a muconatepathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize muconate. In this specific embodiment it can beuseful to increase the synthesis or accumulation of a muconate pathwayproduct to, for example, drive muconate pathway reactions towardmuconate production. Increased synthesis or accumulation can beaccomplished by, for example, overexpression of nucleic acids encodingone or more of the above-described muconate pathway enzymes or proteins.Overexpression of the enzyme or enzymes and/or protein or proteins ofthe muconate pathway can occur, for example, through exogenousexpression of the endogenous gene or genes, or through exogenousexpression of the heterologous gene or genes. Therefore, naturallyoccurring organisms can be readily generated to be non-naturallyoccurring microbial organisms of the invention, for example, producingmuconate, through overexpression of one, two, three, four, five, six,that is, up to all nucleic acids encoding muconate biosynthetic pathwayenzymes or proteins. In addition, a non-naturally occurring organism canbe generated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the muconate biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a muconate biosynthetic pathway onto the microbial organism.Alternatively, encoding nucleic acids can be introduced to produce anintermediate microbial organism having the biosynthetic capability tocatalyze some of the required reactions to confer muconate biosyntheticcapability. For example, a non-naturally occurring microbial organismhaving a muconate biosynthetic pathway can comprise at least twoexogenous nucleic acids encoding desired enzymes or proteins. Thus, itis understood that any combination of two or more enzymes or proteins ofa biosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes or proteins of a biosyntheticpathway can be included in a non-naturally occurring microbial organismof the invention and so forth, as desired, so long as the combination ofenzymes and/or proteins of the desired biosynthetic pathway results inproduction of the corresponding desired product. Similarly, anycombination of four, or more enzymes or proteins of a biosyntheticpathway as disclosed herein can be included in a non-naturally occurringmicrobial organism of the invention, as desired, so long as thecombination of enzymes and/or proteins of the desired biosyntheticpathway results in production of the corresponding desired product.

In addition to the biosynthesis of muconate as described herein, thenon-naturally occurring microbial organisms and methods of the inventionalso can be utilized in various combinations with each other and withother microbial organisms and methods well known in the art to achieveproduct biosynthesis by other routes. For example, one alternative toproduce muconate other than use of the muconate producers is throughaddition of another microbial organism capable of converting a muconatepathway intermediate to muconate. One such procedure includes, forexample, the fermentation of a microbial organism that produces amuconate pathway intermediate. The muconate pathway intermediate canthen be used as a substrate for a second microbial organism thatconverts the muconate pathway intermediate to muconate. The muconatepathway intermediate can be added directly to another culture of thesecond organism or the original culture of the muconate pathwayintermediate producers can be depleted of these microbial organisms by,for example, cell separation, and then subsequent addition of the secondorganism to the fermentation broth can be utilized to produce the finalproduct without intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, muconate. In theseembodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis of muconatecan be accomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, muconatealso can be biosynthetically produced from microbial organisms throughco-culture or co-fermentation using two organisms in the same vessel,where the first microbial organism produces a muconate intermediate andthe second microbial organism converts the intermediate to muconate.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce muconate.

Sources of encoding nucleic acids for a muconate pathway enzyme orprotein can include, for example, any species where the encoded geneproduct is capable of catalyzing the referenced reaction. Such speciesinclude both prokaryotic and eukaryotic organisms including, but notlimited to, bacteria, including archaea and eubacteria, and eukaryotes,including yeast, plant, insect, animal, and mammal, including human.Exemplary species for such sources include, for example, Escherichiacoli, as well as other exemplary species disclosed herein or availableas source organisms for corresponding genes. However, with the completegenome sequence available for now more than 550 species (with more thanhalf of these available on public databases such as the NCBI), including395 microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisitemuconate biosynthetic activity for one or more genes in related ordistant species, including for example, homologues, orthologs, paralogsand nonorthologous gene displacements of known genes, and theinterchange of genetic alterations between organisms is routine and wellknown in the art. Accordingly, the metabolic alterations enablingbiosynthesis of muconate described herein with reference to a particularorganism such as E. coli can be readily applied to other microorganisms,including prokaryotic and eukaryotic organisms alike. Given theteachings and guidance provided herein, those skilled in the art willknow that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In some instances, such as when an alternative muconate biosyntheticpathway exists in an unrelated species, muconate biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesizemuconate.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger andPichia pastoris. E. coli is a particularly useful host organisms sinceit is a well characterized microbial organism suitable for geneticengineering. Other particularly useful host organisms include yeast suchas Saccharomyces cerevisiae.

Methods for constructing and testing the expression levels of anon-naturally occurring muconate-producing host can be performed, forexample, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production ofmuconate can be introduced stably or transiently into a host cell usingtechniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore muconate biosynthetic pathway encoding nucleic acids as exemplifiedherein operably linked to expression control sequences functional in thehost organism. Expression vectors applicable for use in the microbialhost organisms of the invention include, for example, plasmids, phagevectors, viral vectors, episomes and artificial chromosomes, includingvectors and selection sequences or markers operable for stableintegration into a host chromosome. Additionally, the expression vectorscan include one or more selectable marker genes and appropriateexpression control sequences. Selectable marker genes also can beincluded that, for example, provide resistance to antibiotics or toxins,complement auxotrophic deficiencies, or supply critical nutrients not inthe culture media. Expression control sequences can include constitutiveand inducible promoters, transcription enhancers, transcriptionterminators, and the like which are well known in the art. When two ormore exogenous encoding nucleic acids are to be co-expressed, bothnucleic acids can be inserted, for example, into a single expressionvector or in separate expression vectors. For single vector expression,the encoding nucleic acids can be operationally linked to one commonexpression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter.The transformation of exogenous nucleic acid sequences involved in ametabolic or synthetic pathway can be confirmed using methods well knownin the art. Such methods include, for example, nucleic acid analysissuch as Northern blots or polymerase chain reaction (PCR) amplificationof mRNA, or immunoblotting for expression of gene products, or othersuitable analytical methods to test the expression of an introducednucleic acid sequence or its corresponding gene product. It isunderstood by those skilled in the art that the exogenous nucleic acidis expressed in a sufficient amount to produce the desired product, andit is further understood that expression levels can be optimized toobtain sufficient expression using methods well known in the art and asdisclosed herein.

Directed evolution is a powerful approach that involves the introductionof mutations targeted to a specific gene in order to improve and/oralter the properties of an enzyme. Improved and/or altered enzymes canbe identified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (e.g., >10⁴). Iterative rounds of mutagenesis andscreening typically are performed to afford an enzyme with optimizedproperties. Computational algorithms that can help to identify areas ofthe gene for mutagenesis also have been developed and can significantlyreduce the number of enzyme variants that need to be generated andscreened.

Numerous directed evolution technologies have been developed (forreviews, see Hibbert et al., Biomol. Eng 22:11-19 (2005); Huisman andLalonde, In Biocatalysis in the pharmaceutical and biotechnologyindustries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax,Biomol. Eng 22:1-9 (2005); and Sen et al., Appl Biochem. Biotechnol143:212-223 (2007)) to be effective at creating diverse variantlibraries and these methods have been successfully applied to theimprovement of a wide range of properties across many enzyme classes.

Enzyme characteristics that have been improved and/or altered bydirected evolution technologies include, for example,selectivity/specificity—for conversion of non-natural substrates;temperature stability—for robust high temperature processing; pHstability—for bioprocessing under lower or higher pH conditions;substrate or product tolerance—so that high product titers can beachieved; binding (K_(m))—broadens substrate binding to includenon-natural substrates; inhibition (K_(i))—to remove inhibition byproducts, substrates, or key intermediates; activity (kcat)—increasesenzymatic reaction rates to achieve desired flux; expressionlevels—increases protein yields and overall pathway flux; oxygenstability—for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity—for operation of an aerobic enzyme inthe absence of oxygen.

The following exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Any of these can be used to alter/optimize activity of adecarboxylase enzyme.

EpPCR (Pritchard et al., J Theor. Biol 234:497-509 (2005)) introducesrandom point mutations by reducing the fidelity of DNA polymerase in PCRreactions by the addition of Mn²⁺ ions, by biasing dNTP concentrations,or by other conditional variations. The five step cloning process toconfine the mutagenesis to the target gene of interest involves: 1)error-prone PCR amplification of the gene of interest; 2) restrictionenzyme digestion; 3) gel purification of the desired DNA fragment; 4)ligation into a vector; 5) transformation of the gene variants into asuitable host and screening of the library for improved performance.This method can generate multiple mutations in a single genesimultaneously, which can be useful. A high number of mutants can begenerated by EpPCR, so a high-throughput screening assay or a selectionmethod (especially using robotics) is useful to identify those withdesirable characteristics.

Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., NucleicAcids Res 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497(2006)) has many of the same elements as epPCR except a whole circularplasmid is used as the template and random 6-mers with exonucleaseresistant thiophosphate linkages on the last 2 nucleotides are used toamplify the plasmid followed by transformation into cells in which theplasmid is re-circularized at tandem repeats. Adjusting the Mn²⁺concentration can vary the mutation rate somewhat. This technique uses asimple error-prone, single-step method to create a full copy of theplasmid with 3-4 mutations/kbp. No restriction enzyme digestion orspecific primers are required. Additionally, this method is typicallyavailable as a kit.

DNA or Family Shuffling (Stemmer, Proc. Natl. Acad. Sci. U.S.A.91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)) typicallyinvolves digestion of two or more variant genes with nucleases such asDnase I or EndoV to generate a pool of random fragments that arereassembled by cycles of annealing and extension in the presence of DNApolymerase to create a library of chimeric genes. Fragments prime eachother and recombination occurs when one copy primes another copy(template switch). This method can be used with >1 kbp DNA sequences. Inaddition to mutational recombinants created by fragment reassembly, thismethod introduces point mutations in the extension steps at a ratesimilar to error-prone PCR. The method can be used to removedeleterious, random and neutral mutations that might conferantigenicity.

Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol 16:258-261(1998)) entails template priming followed by repeated cycles of 2 stepPCR with denaturation and very short duration of annealing/extension (asshort as 5 sec). Growing fragments anneal to different templates andextend further, which is repeated until full-length sequences are made.Template switching means most resulting fragments have multiple parents.Combinations of low-fidelity polymerases (Taq and Mutazyme) reduceerror-prone biases because of opposite mutational spectra.

In Random Priming Recombination (RPR) random sequence primers are usedto generate many short DNA fragments complementary to different segmentsof the template. (Shao et al., Nucleic Acids Res 26:681-683 (1998)) Basemisincorporation and mispriming via epPCR give point mutations. ShortDNA fragments prime one another based on homology and are recombined andreassembled into full-length by repeated thermocycling. Removal oftemplates prior to this step assures low parental recombinants. Thismethod, like most others, can be performed over multiple iterations toevolve distinct properties. This technology avoids sequence bias, isindependent of gene length, and requires very little parent DNA for theapplication.

In Heteroduplex Recombination linearized plasmid DNA is used to formheteroduplexes that are repaired by mismatch repair. (Volkov et al,Nucleic Acids Res 27:e18 (1999); and Volkov et al., Methods Enzymol.328:456-463 (2000)) The mismatch repair step is at least somewhatmutagenic. Heteroduplexes transform more efficiently than linearhomoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al.,Nat. Biotechnol 19:354-359 (2001)) employs Dnase I fragmentation andsize fractionation of ssDNA. Homologous fragments are hybridized in theabsence of polymerase to a complementary ssDNA scaffold. Any overlappingunhybridized fragment ends are trimmed down by an exonuclease. Gapsbetween fragments are filled in, and then ligated to give a pool offull-length diverse strands hybridized to the scaffold (that contains Uto preclude amplification). The scaffold then is destroyed and isreplaced by a new strand complementary to the diverse strand by PCRamplification. The method involves one strand (scaffold) that is fromonly one parent while the priming fragments derive from other genes; theparent scaffold is selected against. Thus, no reannealing with parentalfragments occurs. Overlapping fragments are trimmed with an exonuclease.Otherwise, this is conceptually similar to DNA shuffling and StEP.Therefore, there should be no siblings, few inactives, and no unshuffledparentals. This technique has advantages in that few or no parentalgenes are created and many more crossovers can result relative tostandard DNA shuffling.

Recombined Extension on Truncated templates (RETT) entails templateswitching of unidirectionally growing strands from primers in thepresence of unidirectional ssDNA fragments used as a pool of templates.(Lee et al., J. Molec. Catalysis 26:119-129 (2003)) No DNA endonucleasesare used. Unidirectional ssDNA is made by DNA polymerase with randomprimers or serial deletion with exonuclease. Unidirectional ssDNA areonly templates and not primers. Random priming and exonucleases don'tintroduce sequence bias as true of enzymatic cleavage of DNAshuffling/RACHITT. RETT can be easier to optimize than StEP because ituses normal PCR conditions instead of very short extensions.Recombination occurs as a component of the PCR steps—no directshuffling. This method can also be more random than StEP due to theabsence of pauses.

In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primersare used to control recombination between molecules; (Bergquist andGibbs, Methods Mol. Biol. 352:191-204 (2007); Bergquist et al., Biomol.Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)) this can beused to control the tendency of other methods such as DNA shuffling toregenerate parental genes. This method can be combined with randommutagenesis (epPCR) of selected gene segments. This can be a good methodto block the reformation of parental sequences. No endonucleases areneeded. By adjusting input concentrations of segments made, one can biastowards a desired backbone. This method allows DNA shuffling fromunrelated parents without restriction enzyme digests and allows a choiceof random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY)creates a combinatorial library with 1 base pair deletions of a gene orgene fragment of interest. (Ostermeier et al., Proc. Natl. Acad. Sci.U.S.A. 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol17:1205-1209 (1999)) Truncations are introduced in opposite direction onpieces of 2 different genes. These are ligated together and the fusionsare cloned. This technique does not require homology between the 2parental genes. When ITCHY is combined with DNA shuffling, the system iscalled SCRATCHY (see below). A major advantage of both is no need forhomology between parental genes; for example, functional fusions betweenan E. coli and a human gene were created via ITCHY. When ITCHY librariesare made, all possible crossovers are captured.

Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs areused to generate truncations. (Lutz et al., Nucleic Acids Res 29:E16(2001)) Relative to ITCHY, THIO-ITCHY can be easier to optimize, providemore reproducibility, and adjustability.

SCRATCHY combines two methods for recombining genes, ITCHY and DNAshuffling. (Lutz et al., Proc. Natl. Acad. Sci. U.S.A. 98:11248-11253(2001)) SCRATCHY combines the best features of ITCHY and DNA shuffling.First, ITCHY is used to create a comprehensive set of fusions betweenfragments of genes in a DNA homology-independent fashion. Thisartificial family is then subjected to a DNA-shuffling step to augmentthe number of crossovers. Computational predictions can be used inoptimization. SCRATCHY is more effective than DNA shuffling whensequence identity is below 80%.

In Random Drift Mutagenesis (RNDM) mutations made via epPCR followed byscreening/selection for those retaining usable activity. (Bergquist etal., Biomol. Eng 22:63-72 (2005)) Then, these are used in DOGS togenerate recombinants with fusions between multiple active mutants orbetween active mutants and some other desirable parent. Designed topromote isolation of neutral mutations; its purpose is to screen forretained catalytic activity whether or not this activity is higher orlower than in the original gene. RNDM is usable in high throughputassays when screening is capable of detecting activity above background.RNDM has been used as a front end to DOGS in generating diversity. Thetechnique imposes a requirement for activity prior to shuffling or othersubsequent steps; neutral drift libraries are indicated to result inhigher/quicker improvements in activity from smaller libraries. Thoughpublished using epPCR, this could be applied to other large-scalemutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis methodthat: 1) generates pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage; this pool isused as a template to 2) extend in the presence of “universal” basessuch as inosine; 3) replication of a inosine-containing complement givesrandom base incorporation and, consequently, mutagenesis. (Wong et al.,Biotechnol J. 3:74-82 (2008); Wong et al., Nucleic Acids Res 32:e26(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)) Using thistechnique it can be possible to generate a large library of mutantswithin 2-3 days using simple methods. This technique is non-directed incomparison to the mutational bias of DNA polymerases. Differences inthis approach makes this technique complementary (or an alternative) toepPCR.

In Synthetic Shuffling, overlapping oligonucleotides are designed toencode “all genetic diversity in targets” and allow a very highdiversity for the shuffled progeny. (Ness et al., Nat. Biotechnol20:1251-1255 (2002)) In this technique, one can design the fragments tobe shuffled. This aids in increasing the resulting diversity of theprogeny. One can design sequence/codon biases to make more distantlyrelated sequences recombine at rates approaching those observed withmore closely related sequences. Additionally, the technique does notrequire physically possessing the template genes.

Nucleotide Exchange and Excision Technology NexT exploits a combinationof dUTP incorporation followed by treatment with uracil DNA glycosylaseand then piperidine to perform endpoint DNA fragmentation. (Muller etal., Nucleic Acids Res 33:e117 (2005)) The gene is reassembled usinginternal PCR primer extension with proofreading polymerase. The sizesfor shuffling are directly controllable using varying dUPT::dTTP ratios.This is an end point reaction using simple methods for uracilincorporation and cleavage. Other nucleotide analogs, such as8-oxo-guanine, can be used with this method. Additionally, the techniqueworks well with very short fragments (86 bp) and has a low error rate.The chemical cleavage of DNA used in this technique results in very fewunshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC) alinker is used to facilitate fusion between two distantly/unrelatedgenes. Nuclease treatment is used to generate a range of chimerasbetween the two genes. These fusions result in libraries ofsingle-crossover hybrids. (Sieber et al., Nat. Biotechnol 19:456-460(2001)) This produces a limited type of shuffling and a separate processis required for mutagenesis. In addition, since no homology is neededthis technique can create a library of chimeras with varying fractionsof each of the two unrelated parent genes. SHIPREC was tested with aheme-binding domain of a bacterial CP450 fused to N-terminal regions ofa mammalian CP450; this produced mammalian activity in a more solubleenzyme.

In Gene Site Saturation Mutagenesis™ (GSSM™) the starting materials area supercoiled dsDNA plasmid containing an insert and two primers whichare degenerate at the desired site of mutations. (Kretz et al., MethodsEnzymol. 388:3-11 (2004)) Primers carrying the mutation of interest,anneal to the same sequence on opposite strands of DNA. The mutation istypically in the middle of the primer and flanked on each side by ˜20nucleotides of correct sequence. The sequence in the primer is NNN orNNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A, C). Afterextension, DpnI is used to digest dam-methylated DNA to eliminate thewild-type template. This technique explores all possible amino acidsubstitutions at a given locus (i.e., one codon). The techniquefacilitates the generation of all possible replacements at a single-sitewith no nonsense codons and results in equal to near-equalrepresentation of most possible alleles. This technique does not requireprior knowledge of the structure, mechanism, or domains of the targetenzyme. If followed by shuffling or Gene Reassembly, this technologycreates a diverse library of recombinants containing all possiblecombinations of single-site up-mutations. The utility of this technologycombination has been demonstrated for the successful evolution of over50 different enzymes, and also for more than one property in a givenenzyme.

Combinatorial Cassette Mutagenesis (CCM) involves the use of shortoligonucleotide cassettes to replace limited regions with a large numberof possible amino acid sequence alterations. (Reidhaar-Olson et al.Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science241:53-57 (1988)) Simultaneous substitutions at two or three sites arepossible using this technique. Additionally, the method tests a largemultiplicity of possible sequence changes at a limited range of sites.This technique has been used to explore the information content of thelambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentiallysimilar to CCM except it is employed as part of a larger program: 1) Useof epPCR at high mutation rate to 2) ID hot spots and hot regions andthen 3) extension by CMCM to cover a defined region of protein sequencespace. (Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001).)As with CCM, this method can test virtually all possible alterationsover a target region. If used along with methods to create randommutations and shuffled genes, it provides an excellent means ofgenerating diverse, shuffled proteins. This approach was successful inincreasing, by 51-fold, the enantioselectivity of an enzyme.

In the Mutator Strains technique conditional is mutator plasmids allowincreases of 20- to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required. (Selifonova et al., Appl Environ Microbiol67:3645-3649 (2001)) This technology is based on a plasmid-derived mutD5gene, which encodes a mutant subunit of DNA polymerase III. This subunitbinds to endogenous DNA polymerase III and compromises the proofreadingability of polymerase III in any strain that harbors the plasmid. Abroad-spectrum of base substitutions and frameshift mutations occur. Inorder for effective use, the mutator plasmid should be removed once thedesired phenotype is achieved; this is accomplished through atemperature sensitive origin of replication, which allows for plasmidcuring at 41° C. It should be noted that mutator strains have beenexplored for quite some time (e.g., see Low et al., J. Mol. Biol.260:359-3680 (1996)). In this technique very high spontaneous mutationrates are observed. The conditional property minimizes non-desiredbackground mutations. This technology could be combined with adaptiveevolution to enhance mutagenesis rates and more rapidly achieve desiredphenotypes.

“Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis methodthat assesses and optimizes combinatorial mutations of selected aminoacids.” (Rajpal et al., Proc Natl Acad Sci U.S.A. 102:8466-8471 (2005))Rather than saturating each site with all possible amino acid changes, aset of nine is chosen to cover the range of amino acid R-groupchemistry. Fewer changes per site allows multiple sites to be subjectedto this type of mutagenesis. A >800-fold increase in binding affinityfor an antibody from low nanomolar to picomolar has been achievedthrough this method. This is a rational approach to minimize the numberof random combinations and can increase the ability to find improvedtraits by greatly decreasing the numbers of clones to be screened. Thishas been applied to antibody engineering, specifically to increase thebinding affinity and/or reduce dissociation. The technique can becombined with either screens or selections.

Gene Reassembly is a DNA shuffling method that can be applied tomultiple genes at one time or to creating a large library of chimeras(multiple mutations) of a single gene. (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation) Typically this technologyis used in combination with ultra-high-throughput screening to query therepresented sequence space for desired improvements. This techniqueallows multiple gene recombination independent of homology. The exactnumber and position of cross-over events can be pre-determined usingfragments designed via bioinformatic analysis. This technology leads toa very high level of diversity with virtually no parental genereformation and a low level of inactive genes. Combined with GSSM™, alarge range of mutations can be tested for improved activity. The methodallows “blending” and “fine tuning” of DNA shuffling, e.g. codon usagecan be optimized.

In Silico Protein Design Automation (PDA) is an optimization algorithmthat anchors the structurally defined protein backbone possessing aparticular fold, and searches sequence space for amino acidsubstitutions that can stabilize the fold and overall proteinenergetics. (Hayes et al., Proc Natl Acad Sci U.S.A. 99:15926-15931(2002)) This technology uses in silico structure-based entropypredictions in order to search for structural tolerance toward proteinamino acid variations. Statistical mechanics is applied to calculatecoupling interactions at each position. Structural tolerance towardamino acid substitution is a measure of coupling. Ultimately, thistechnology is designed to yield desired modifications of proteinproperties while maintaining the integrity of structuralcharacteristics. The method computationally assesses and allowsfiltering of a very large number of possible sequence variants (10⁵⁰).The choice of sequence variants to test is related to predictions basedon the most favorable thermodynamics. Ostensibly only stability orproperties that are linked to stability can be effectively addressedwith this technology. The method has been successfully used in sometherapeutic proteins, especially in engineering immunoglobulins. Insilico predictions avoid testing extraordinarily large numbers ofpotential variants. Predictions based on existing three-dimensionalstructures are more likely to succeed than predictions based onhypothetical structures. This technology can readily predict and allowtargeted screening of multiple simultaneous mutations, something notpossible with purely experimental technologies due to exponentialincreases in numbers.

Iterative Saturation Mutagenesis (ISM) involves: 1) use knowledge ofstructure/function to choose a likely site for enzyme improvement; 2)saturation mutagenesis at chosen site using Stratagene QuikChange (orother suitable means); 3) screen/select for desired properties; and 4)with improved clone(s), start over at another site and continuerepeating. (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz etal., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)) This is a provenmethodology, which assures all possible replacements at a given positionare made for screening/selection.

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques.

The present invention provides a method for producing muconate thatincludes culturing a non-naturally occurring microbial organism having amuconate pathway. The pathway includes at least one exogenous nucleicacid encoding a muconate pathway enzyme expressed in a sufficient amountto produce muconate, under conditions and for a sufficient period oftime to produce muconate. The muconate pathway includes an enzymeselected from the group consisting of a beta-ketothiolase, abeta-ketoadipyl-CoA hydrolase, a beta-ketoadipyl-CoA transferase, abeta-ketoadipyl-CoA ligase, a 2-fumarylacetate reductase, a2-fumarylacetate dehydrogenase, a trans-3-hydroxy-4-hexendioatedehydratase, a 2-fumarylacetate aminotransferase, a 2-fumarylacetateaminating oxidoreductase, a trans-3-amino-4-hexenoate deaminase, abeta-ketoadipate enol-lactone hydrolase, a muconolactone isomerase, amuconate cycloisomerase, a beta-ketoadipyl-CoA dehydrogenase, a3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA transferase, a2,3-dehydroadipyl-CoA hydrolase, a 2,3-dehydroadipyl-CoA ligase, amuconate reductase, a 2-maleylacetate reductase, a 2-maleylacetatedehydrogenase, a cis-3-hydroxy-4-hexendioate dehydratase, a2-maleylacetate aminoatransferase, a 2-maleylacetate aminatingoxidoreductase, a cis-3-amino-4-hexendioate deaminase, and a muconatecis/trans isomerase.

In some embodiments, the muconate pathway includes, a set of muconatepathway enzymes such as those exemplified in FIG. 2; the set of muconatepathway enzymes are selected from the group consisting of:

A) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase, andbeta-ketoadipyl-CoA ligase, (3) beta-ketoadipate enol-lactone hydrolase,(4) muconolactone isomerase, (5) muconate cycloisomerase, and (6)muconate cis/trans isomerase;

B) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase andbeta-ketoadipyl-CoA ligase, (3) 2-maleylacetate reductase, (4)2-maleylacetate dehydrogenase, (5) cis-3-hydroxy-4-hexendioatedehydratase, and (6) muconate cis/trans isomerase;

C) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase andbeta-ketoadipyl-CoA ligase, (3) 2-maleylacetate reductase, (4) an enzymeselected from 2-maleylacetate aminotransferase and 2-maleylacetateaminating oxidoreductase, (5) cis-3-amino-4-hexenoate deaminase, and (6)muconate cis/trans isomerase;

D) (1) beta-ketothiolase, (2) beta-ketoadipyl-CoA dehydrogenase, (3)3-hydroxyadipyl-CoA dehydratase, (4) an enzyme selected from2,3-dehydroadipyl-CoA transferase, 2,3-dehydroadipyl-CoA hydrolase and2,3-dehydroadipyl-CoA ligase, and (5) muconate reductase;

E) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase andbeta-ketoadipyl-CoA ligase, (3) 2-fumarylacetate reductase, (4)2-fumarylacetate dehydrogenase, and (5) trans-3-hydroxy-4-hexendioatedehydratase;

F) (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase andbeta-ketoadipyl-CoA ligase, (3) 2-fumarylacetate reductase, (4) anenzyme selected from 2-fumarylacetate aminotransferase and2-fumarylacetate aminating oxidoreductase, and (5)trans-3-amino-4-hexenoate deaminase.

In some embodiments, the present invention provides a method forproducing muconate that includes culturing a non-naturally occurringmicrobial organism having a muconate pathway. The pathway comprising atleast one exogenous nucleic acid encoding a muconate pathway enzymeexpressed in a sufficient amount to produce muconate, under conditionsand for a sufficient period of time to produce muconate. The muconatepathway includes a 4-hydroxy-2-ketovalerate aldolase, a 2-oxopentenoatehydratase, a 4-oxalocrotonate dehydrogenase, a 2-hydroxy-4-hexenedioatedehydratase, a 4-hydroxy-2-oxohexanedioate oxidoreductase, a2,4-dihydroxyadipate dehydratase (acting on 2-hydroxy), a2,4-dihydroxyadipate dehydratase (acting on 4-hydroxyl group) and a3-hydroxy-4-hexenedioate dehydratase.

In some embodiments, the muconate pathway includes, a set of muconatepathway enzymes such as those exemplified in FIG. 3; the set of muconatepathway enzymes are selected from the group consisting of:

A) (1) 4-hydroxy-2-ketovalerate aldolase, (2) 2-oxopentenoate hydratase,(3) 4-oxalocrotonate dehydrogenase, (4) 2-hydroxy-4-hexenedioatedehydratase;

B) (1) 4-hydroxy-2-ketovalerate aldolase, (2)4-hydroxy-2-oxohexanedioate oxidoreductase, (3) 2,4-dihydroxyadipatedehydratase (acting on 2-hydroxy), (4) 3-hydroxy-4-hexenedioatedehydratase; and

C) (1) 4-hydroxy-2-ketovalerate aldolase, (2)4-hydroxy-2-oxohexanedioate oxidoreductase, (3) 2,4-dihydroxyadipatedehydratase (acting on 4-hydroxyl group), (4) 2-hydroxy-4-hexenedioatedehydratase.

In some embodiments, the present invention provides a method forproducing muconate that includes culturing a non-naturally occurringmicrobial organism having a muconate pathway. The pathway includes atleast one exogenous nucleic acid encoding a muconate pathway enzymeexpressed in a sufficient amount to produce muconate, under conditionsand for a sufficient period of time to produce muconate. The muconatepathway includes an enzyme selected from the group consisting of an HODHaldolase, an OHED hydratase, an OHED decarboxylase, an HODHformate-lyase, an HODH dehydrogenase, an OHED formate-lyase, an OHEDdehydrogenase, a 6-OHE dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase,a 2,3-dehydroadipyl-CoA hydrolase, a 2,3-dehydroadipyl-CoA transferase,a 2,3-dehydroadipyl-CoA ligase, and a muconate reductase.

In some embodiments, the muconate pathway includes, a set of muconatepathway enzymes such as those exemplified in FIG. 4; the set of muconatepathway enzymes are selected from the group consisting of:

A) (1) HODH aldolase, (2) OHED hydratase, (3) OHED decarboxylase, (4)6-OHE dehydrogenase, and (5) muconate reductase;

B) (1) HODH aldolase, (2) OHED hydratase, (3) an enzyme selected fromOHED formate-lyase and OHED dehydrogenase, (4) an enzyme selected from2,3-dehydroadipyl-CoA hydrolase, 2,3-dehydroadipyl-CoA transferase and2,3-dehydroadipyl-CoA ligase, and (5) muconate reductase; and

C) (1) HODH aldolase, (2) an enzyme selected from HODH formate-lyase andHODH dehydrogenase, (3) 3-hydroxyadipyl-CoA dehydratase, (4) an enzymeselected from 2,3-dehydroadipyl-CoA hydrolase, 2,3-dehydroadipyl-CoAtransferase and 2,3-dehydroadipyl-CoA ligase, and (5) muconatereductase.

In some embodiments, the present invention provides a method forproducing muconate that includes culturing a non-naturally occurringmicrobial organism having a muconate pathway. The pathway includes atleast one exogenous nucleic acid encoding a muconate pathway enzymeexpressed in a sufficient amount to produce muconate, under conditionsand for a sufficient period of time to produce muconate. The muconatepathway includes an enzyme selected from the group consisting of alysine aminotransferase, a lysine aminating oxidoreductase, a2-aminoadipate semialdehyde dehydrogenase, a 2-aminoadipate deaminase, amuconate reductase, a lysine-2,3-aminomutase, a 3,6-diaminohexanoateaminotransferase, a 3,6-diaminohexanoate aminating oxidoreductase, a3-aminoadipate semialdehyde dehydrogenase, and a 3-aminoadipatedeaminase.

In some embodiments, the muconate pathway includes, a set of muconatepathway enzymes such as those exemplified in FIG. 5; the set of muconatepathway enzymes are selected from the group consisting of:

A) (1) lysine aminotransferase, (2) lysine aminating oxidoreductase, (3)2-aminoadipate semialdehyde dehydrogenase, (4) 2-aminoadipate deaminase,and (5) muconate reductase

B) (1) lysine-2,3-aminomutase, (2) 3,6-diaminohexanoateaminotransferase, (3) 3,6-diaminohexanoate aminating oxidoreductase, (4)3-aminoadipate semialdehyde dehydrogenase, (5) 3-aminoadipate deaminase,and (6) muconate reductase.

In some embodiments, the foregoing non-naturally occurring microbialorganism can be cultured in a substantially anaerobic culture medium.

Suitable purification and/or assays to test for the production ofmuconate can be performed using well known methods. Suitable replicatessuch as triplicate cultures can be grown for each engineered strain tobe tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy) or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art. Forexample, a spectrophotometric assay for succinyl-CoA:3-ketoacid-CoAtransferase (FIG. 2, Step B) entails measuring the change in theabsorbance corresponding to the product CoA molecule (i.e.,succinyl-CoA) in the presence of the enzyme extract when supplied withsuccinate and β-ketoadipyl-CoA (Corthesy-Theulaz et al., J Biol Chem.,272(41) (1997)). Succinyl-CoA can alternatively be measured in thepresence of excess hydroxylamine by complexing the succinohydroxamicacid formed to ferric salts as referred to in (Corthesy-Theulaz et al.,J. Biol Chem. 272(41) (1997)). The specific activity of muconatereductase can be assayed in the reductive direction using a colorimetricassay adapted from the literature (Durre et al., FEMS Microbiol. Rev.17:251-262 (1995); Palosaari et al., J. Bacteriol. 170:2971-2976 (1988);Welch et al., Arch. Biochem. Biophys. 273:309-318 (1989)). In thisassay, the substrates muconate and NADH are added to cell extracts in abuffered solution, and the oxidation of NADH is followed by readingabsorbance at 340 nM at regular intervals. The resulting slope of thereduction in absorbance at 340 nM per minute, along with the molarextinction coefficient of NADH at 340 nM (6000) and the proteinconcentration of the extract, can be used to determine the specificactivity of muconate reductase.

The muconate can be separated from other components in the culture usinga variety of methods well known in the art, as briefly described aboveSuch separation methods include, for example, extraction procedures aswell as methods that include continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the muconate producers can be cultured forthe biosynthetic production of muconate.

For the production of muconate, the recombinant strains are cultured ina medium with carbon source and other essential nutrients. It is highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic conditions can be applied byperforating the septum with a small hole for limited aeration. Exemplaryanaerobic conditions have been described previously and are well-knownin the art. Exemplary aerobic and anaerobic conditions are described,for example, in U.S. patent application Ser. No. 11/891,602, filed Aug.10, 2007. Fermentations can be performed in a batch, fed-batch orcontinuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.NOTE—Ideally this process would operate at low pH using an organismsthat tolerates pH levels in the range 2-4.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of muconate.

In addition to renewable feedstocks such as those exemplified above, themuconate microbial organisms of the invention also can be modified forgrowth on syngas as its source of carbon. In this specific embodiment,one or more proteins or enzymes are expressed in the muconate producingorganisms to provide a metabolic pathway for utilization of syngas orother gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a muconate pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the Wood-Ljungdahl enzymes or proteins absent in the hostorganism. Therefore, introduction of one or more encoding nucleic acidsinto the microbial organisms of the invention such that the modifiedorganism contains the complete Wood-Ljungdahl pathway will confer syngasutilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, muconate and any ofthe intermediate metabolites in the muconate pathway. All that isrequired is to engineer in one or more of the required enzyme or proteinactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of themuconate biosynthetic pathways. Accordingly, the invention provides anon-naturally occurring microbial organism that produces and/or secretesmuconate when grown on a carbohydrate or other carbon source andproduces and/or secretes any of the intermediate metabolites shown inthe muconate pathway when grown on a carbohydrate or other carbonsource. The muconate producing microbial organisms of the invention caninitiate synthesis from an intermediate, such as any of theintermediates shown in FIGS. 2-5.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a muconatepathway enzyme or protein in sufficient amounts to produce muconate. Itis understood that the microbial organisms of the invention are culturedunder conditions sufficient to produce muconate. Following the teachingsand guidance provided herein, the non-naturally occurring microbialorganisms of the invention can achieve biosynthesis of muconateresulting in intracellular concentrations between about 0.1-200 mM ormore. Generally, the intracellular concentration of muconate is betweenabout 3-150 mM, particularly between about 5-200 mM and moreparticularly between about 8-150 mM, including about 10 mM, 50 mM, 75mM, 100 mM, or more. Intracellular concentrations between and above eachof these exemplary ranges also can be achieved from the non-naturallyoccurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007. Any of theseconditions can be employed with the non-naturally occurring microbialorganisms as well as other anaerobic conditions well known in the art.Under such anaerobic conditions, the muconate producers can synthesizemuconate at intracellular concentrations of 5-10 mM or more as well asall other concentrations exemplified herein. It is understood that, eventhough the above description refers to intracellular concentrations,muconate producing microbial organisms can produce muconateintracellularly and/or secrete the product into the culture medium.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of muconate includes anaerobic culture or fermentationconditions. In certain embodiments, the non-naturally occurringmicrobial organisms of the invention can be sustained, cultured orfermented under anaerobic or substantially anaerobic conditions.Briefly, anaerobic conditions refers to an environment devoid of oxygen.Substantially anaerobic conditions include, for example, a culture,batch fermentation or continuous fermentation such that the dissolvedoxygen concentration in the medium remains between 0 and 10% ofsaturation. Substantially anaerobic conditions also includes growing orresting cells in liquid medium or on solid agar inside a sealed chambermaintained with an atmosphere of less than 1% oxygen. The percent ofoxygen can be maintained by, for example, sparging the culture with anN₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of muconate. Exemplary growth proceduresinclude, for example, fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation, or continuousfermentation and continuous separation. All of these processes are wellknown in the art. Fermentation procedures are particularly useful forthe biosynthetic production of commercial quantities of muconate.Generally, and as with non-continuous culture procedures, the continuousand/or near-continuous production of muconate will include culturing anon-naturally occurring muconate producing organism of the invention insufficient nutrients and medium to sustain and/or nearly sustain growthin an exponential phase. Continuous culture under such conditions can beinclude, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 ormore weeks and up to several months. Alternatively, organisms of theinvention can be cultured for hours, if suitable for a particularapplication. It is to be understood that the continuous and/ornear-continuous culture conditions also can include all time intervalsin between these exemplary periods. It is further understood that thetime of culturing the microbial organism of the invention is for asufficient period of time to produce a sufficient amount of product fora desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of muconate can be utilized in, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

In addition to the above fermentation procedures using the muconateproducers of the invention for continuous production of substantialquantities of muconate, the muconate producers also can be, for example,simultaneously subjected to chemical synthesis procedures to convert theproduct to other compounds or the product can be separated from thefermentation culture and sequentially subjected to chemical conversionto convert the product to other compounds, if desired.

In addition to the above procedures, growth condition for achievingbiosynthesis of muconate can include the addition of an osmoprotectantto the culturing conditions. In certain embodiments, the non-naturallyoccurring microbial organisms of the invention can be sustained,cultured or fermented as described above in the presence of anosmoprotectant. Briefly, an osmoprotectant means a compound that acts asan osmolyte and helps a microbial organism as described herein surviveosmotic stress. Osmoprotectants include, but are not limited to,betaines, amino acids, and the sugar trehalose. Non-limiting examples ofsuch are glycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine andectoine. In one aspect, the osmoprotectant is glycine betaine. It isunderstood to one of ordinary skill in the art that the amount and typeof osmoprotectant suitable for protecting a microbial organism describedherein from osmotic stress will depend on the microbial organism used.The amount of osmoprotectant in the culturing conditions can be, forexample, no more than about 0.1 mM, no more than about 0.5 mM, no morethan about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM,no more than about 2.5 mM, no more than about 3.0 mM, no more than about5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no morethan about 50 mM, no more than about 100 mM or no more than about 500mM.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of muconate.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat enable an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Demonstration of Enzyme Activity for Condensing Succinyl-CoAand Acetyl-CoA to Form β-Ketoadipyl-CoA

This Example shows the identification of enzymes for the formation ofbeta-ketoadipyl-CoA from succinyl-CoA and acetyl-CoA.

Several β-ketothiolase enzymes have been shown to break β-ketoadipyl-CoAinto acetyl-CoA and succinyl-CoA. For example, the gene products encodedby pcaF in Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol.184(1): 207-15 (2002)), phaD in Pseudomonas putida U (Olivera et al.,Proc Natl Acad Sci USA, 95(11), 6419-24 (1998)), paaE in Pseudomonasfluorescens ST (Di Gennaro et al., Arch Microbiol, 188(2), 117-25(2007)), and paaJ from E. coli (Nogales et al., Microbiology 153(Pt 2),357-65 (2007)) catalyze the conversion of 3-oxoadipyl-CoA intosuccinyl-CoA and acetyl-CoA during the degradation of aromatic compoundssuch as phenylacetate or styrene. To confirm that β-ketothiolase enzymesexhibit condensation activity, several thiolases (Table 53) were clonedinto a derivative of pZE13 (Lutz et al., Nucleic Acids Res, 29(18),3873-81 (2001)), which results in the clones having a carboxy-terminal6×His tag.

TABLE 53 Cloned Thiolases Enzyme Species Template Gene Length 5′ PRIMER3′ PRIMER beta- Ralstonia bktB 1185 ATGACGCGTG GATACGCTCGA ketothiolaseeutropha H16 AAGTGGTAGT AGATGGCGG GGTAAG 2- Mus musculus ACAT1 1215ATGGAAGTAA CAGCTTCTCAAT Methylacetoacetyl- GATGCCTGGA CAGCAGGGC CoAACGAAG Thiolase (branched chain?) 2- Pseudomonas fadAx 1194 ATGACCCTCGGTACAGGCATTC Methylacetoacetyl- putida (KT2440) CCAATGACCC AACAGCCATGGCoA Thiolase (branched chain?) beta- Caenorhabditis kat-1 1167ATGAACAAAC TAATTTCTGGAT ketothiolase elegans ATGCTTTCATC AACCATTCCACTGTCG TGAGC beta- Escherichia coli paaJ 1206 ATGCGTGAAG AACACGCTCCAketothiolase CCTTTATTTGT GAATCATGGCG NP_415915.1 GACG beta- PseudomonasphaD 1221 ATGAATGAAC GAGGCGCTCGA ketothiolase putida (KT2440) CGACCCACGCTGATCATGG AAN68887.1 C beta- Clostridium thiA 1179 ATGAAAGAAGGCACTTTTCTAG ketothiolase acetobutylicum TTGTAATAGCT CAATATTGCTGTNP_349476.1 ATCC 824 AGTGCAGTAA TCC GAAC beta- Clostridium thiB 1179ATGAGAGATG GTCTCTTTCAAC ketothiolase acetobutylicum TAGTAATAGTTACGAGAGCTGT NP_149242.1 ATCC 824 AAGTGCTGTA TCCC AGAACTG 3-oxoadipyl-Candida albicans POT98 1182 ATGTTCAAGA CTCGTTAGCAAA CoA thiolase SC5314AATCAGCTAA CAAGGCAGCG TGATATTGTTG 3-oxoadipyl- Candela albicans POT11227 ATGGATAGAT TTCCTTAATCAA CoA thiolase SC5314 TAAATCAATT TATGGAGGCAGAAGTGGTCAA CAC TTAAAACC 3-oxoadipyl- Candida albicans POT2 1233ATGTCATCCA TTCTCTAACCAA CoA thiolase SC5314 AACAACAATA AACAGAAGCAGCTTGAAGAAG CACC beta- Pseudomonas pcaF 1206 ATGAGCCGCG GACCCGCTCGATketoadipyl aeruginosa PAO1 AGGTATTCAT GGCCAG CoA thiolase CTG pcaFacyl-CoA Pseudomonas bkt 1206 ATGCTCGATG TCGGCAGCGCTC thiolaseaeruginosa PAO1 CCTATATCTAC GATCAC GCC 3-oxoadipyl- Pseudomonas pcaF1203 ATGCACGACG AACCCGCTCGAT CoA thiolase putida (KT2440) TATTCATCTGTGGCCAAC GACG 3-oxoadipyl- Burkholderia bkt 1203 ATGACCGACG CACGCGTTCGATCoA thiolase ambifaria AMMD CCTACATCTGC CGCGATC G beta- Ascaris suum bkt1242 ATGGCCACCT CAATTTCTCGAT ketothiolase CAAGACTTGT GACCATTCCACC CTGCEnzyme ORF SEQ beta-atgacgcgtgaagtggtagtggtaagcggtgtccgtaccgcgatcgggacctttggcg ketothiolasegcagcctgaaggatgtggcaccggcggagctgggcgcactggtggtgcgcgaggcgctggcgcgcgcgcaggtgtcgggcgacgatgtcggccacgtggtattcggcaacgtgatccagaccgagccgcgcgacatgtatctgggccgcgtcgcggccgtcaacggcggggtgacgatcaacgcccccgcgctgaccgtgaaccgcctgtgcggctcgggcctgcaggccattgtcagcgccgcgcagaccatcctgctgggcgataccgacgtcgccatcggcggcggcgcggaaagcatgagccgcgcaccgtacctggcgccggcagcgcgctggggcgcacgcatgggcgacgccggcctggtcgacatgatgctgggtgcgctgcacgatcccttccatcgcatccacatgggcgtgaccgccgagaatgtcgccaaggaatacgacatctcgcgcgcgcagcaggacgaggccgcgctggaatcgcaccgccgcgcttcggcagcgatcaaggccggctacttcaaggaccagatcgtcccggtggtgagcaagggccgcaagggcgacgtgaccttcgacaccgacgagcacgtgcgccatgacgccaccatcgacgacatgaccaagctcaggccggtcttcgtcaaggaaaacggcacggtcacggccggcaatgcctcgggcctgaacgacgccgccgccgcggtggtgatgatggagcgcgccgaagccgagcgccgcggcctgaagccgctggcccgcctggtgtcgtacggccatgccggcgtggacccgaaggccatgggcatcggcccggtgccggcgacgaagatcgcgctggagcgcgccggcctgcaggtgtcggacctggacgtgatcgaagccaacgaagcctttgccgcacaggcgtgcgccgtgaccaaggcgctcggtctggacccggccaaggttaacccgaacggctcgggcatctcgctgggccacccgatcggcgccaccggtgccctgatcacggtgaaggcgctgcatgagctgaaccgcgtgcagggccgctacgcgctggtgacgatgtgcatcggcggcgggcagggcattgccgccatcttcgagcgtatctga 2-atggaagtaagatgcctggaacgaagttatgcatccaaacccactttgaatgaagtggttMethylacetoacetyl-atagtaagtgctataagaactcccattggatccttcctgggcagccttgcctctcagccg CoAgccactaaacttggtactgctgcaattcagggagccattgagaaggcagggattccaaa Thiolaseagaagaagtgaaggaagtctacatgggcaatgtcatccaagggggtgaaggacagg (branchedcccctaccaggcaagcaacactgggcgcaggtttacctatttccactccatgcaccaca chain?)gtaaacaaggtttgtgcttcaggaatgaaagccatcatgatggcctctcaaagtcttatgtgtggacatcaggatgtgatggtggcaggcgggatggagagcatgtccaatgtcccatacgtaatgagcagaggagcaacaccatatggtggggtaaaacttgaagacctgattgtaaaagacgggctaactgatgtctacaataaaattcatatgggtaactgtgctgagaatactgcaaagaagatgaatatctcacggcaggaacaggatacgtacgctctcagctcttacaccagaagtaaagaagcgtgggacgcagggaagtttgccagtgagattactcccatcaccatctcagtgaaaggtaaaccagatgtggtggtgaaagaagatgaagaatacaagcgtgttgactttagtaaagtgccaaagctcaagaccgtgttccagaaagaaaatggcacaataacagctgccaatgccagcacactgaacgatggagcagctgctctggttctcatgactgcagaggcagcccagaggctcaatgttaagccattggcacgaattgcagcatttgctgatgctgccgtagaccccattgattttccacttgcgcctgcatatgccgtacctaaggttcttaaatatgcaggactgaaaaaagaagacattgccatgtgggaagtaaatgaagcattcagtgtggttgtgctagccaacattaaaatgctggagattgacccccaaaaagtaaatatccacggaggagctgtttctctgggccatccaattgggatgtctggagcccggattgttgttcatatggctcatgccctgaagccaggagagttcggtctggctagtatttgcaacggaggaggaggtgcttccgccctgctgattgagaagctgtag 2-atgaccctcgccaatgaccccatcgttatcgtcagcgccgtgcgcacgcccatgggcgMethylacetoacetyl-ggttgcagggcgacctcaagagcctgactgcgccgcaactgggcagcgccgccattc CoAgtgctgccgtggaacgggccggcatcgatgccgccggtgtcgagcaggtactgttcg Thiolasegctgcgtgctgccggccggccagggccaggcaccggcacgccaggccgcgctggg (branchedcgccgggctggacaagacaccacctgcaccaccctgaacaagatgtgcggctcgg chain?)gtatgcaagccgcgatcatggcccatgacctgctgctggccggcaccgcagacgtggtagtggcgggtggcatggaaagcatgaccaacgcgccgtacctgctggacaaagcccgtggcggctaccgcatgggccacggcaagatcatcgaccacatgttcatggacggtctcgaagacgcctacgacaaaggccgcctgatgggtacctttgccgaggactgtgcccaggccaatgccttcagccgcgaggcccaggaccagttcgccatcgcctcgctgacccgagcgcaggaagccatcagcagcggccgttttgccgccgagatcgtgccggtggaagtcaccgagggcaaggaaaagcgcgtcatcaaggatgacgagcagccgcccaaggcgcgtctggacaagattgcgcagctcaaaccggcgtttcgtgaaggcggcaccgtgacggcggccaacgccagttcgatttccgacggcgctgcggcgctggtactgatgcgccgctccgaggccgacaaacgtggcctcaagccattggccgtcatccacggccacgccgcctttgccgacaccccggcgctgttcccgaccgccccgatcggcgcgatcgacaaactgatgaaacgcaccggctggaacctggccgaagtcgacctgttcgagatcaacgaggccttcgccgtggtcaccctggcggccatgaaacacctcgacctgccacacgacaaggtcaatatccacggcggcgcctgcgccctcggtcacccgatcggcgcttctggcgcacgtattctggtcaccctgttgtcggccttgcgccagaacaatctgcgtcggggtgtggcggccatctgcatcggcggtggcgaggccacggccatggctgttgaatgcctgtactga beta-atgaacaaacatgctttcatcgtcggagccgcccgtacacctattggatcatttcgttcttcketothiolasetctctcttcggtaactgctccagagctcgcctcggttgccatcaaagcagcattggagcgtggagcagtgaagccgagttcaattcaggaggtgttccttggtcaagtctgtcaagcaaatgctggtcaagctcccgctcgtcaagcagctcttggagccggactcgatctttcggttgctgttaccaccgtcaataaagtgtgctcttctgggctgaaagcaatcattcttgctgcccagcaaattcaaaccggtcatcaagattttgccattggcggaggaatggagagcatgtcacaagtaccattttatgttcaaagaggagagatcccatatggtggatttcaagtgattgatggaatcgtcaaagacggactgaccgatgcttatgataaagttcacatgggaaactgcggagagaagacttcaaaagaaatgggaattacacgtaaagaccaagacgaatatgctatcaacagctacaaaaagtcagctaaagcatgggagaatggaaatatcggaccagaagtggtgccagtgaacgtcaaatcaaagaagggagtcacgattgttgataaagatgaagagttcacaaaagtcaatttcgacaagttcacctcgctgagaactgttttccagaaagacggaactatcactgctgctaatgcttcaacattgaacgacggtgcagctgctgtcattgttgcctcacaggaagcagtttccgagcaaagcttaaagcctctggcccgaattttggcttatggagatgccgccacgcacccactcgatttcgctgtagcaccaactttgatgttcccaaaaattcttgaaagagcaggagtgaagcaatcagatgttgctcaatgggaagttaatgaagccttctcatgtgttccccttgctttcatcaaaaaactaggagtcgatccatcccttgtgaacccacatggaggagctgtttcaattggtcaccccatcggaatgtccggagcccgcctcatcactcatcttgtgcacacactcaaaagtggccaaatcggagttgctgccatttgcaatggaggtggtggctcaagtggaatggttatccagaaattataa beta-atgcgtgaagcctttatttgtgacggaattcgtacgccaattggtcgctacggcggggcaketothiolasettatcaagtgttcgggctgatgatctggctgctatccctttgcgggaactgctggtgcgaaNP_415915.1 acccgcgtctcgatgcggagtgtatcgatgatgtgatcctcggctgtgctaatcaggcgggagaagataaccgtaacgtagcccggatggcgactttactggcggggctgccgcagagtgtttccggcacaaccattaaccgcttgtgtggttccgggctggacgcactggggtttgccgcacgggcgattaaagcgggcgatggcgatttgctgatcgccggtggcgtggagtcaatgtcacgggcaccgtttgttatgggcaaggcagccagtgcattttctcgtcaggctgagatgttcgataccactattggctggcgatttgtgaacccgctcatggctcagcaatttggaactgacagcatgccggaaacggcagagaatgtagctgaactgttaaaaatctcacgagaagatcaagatagttttgcgctacgcagtcagcaacgtacggcaaaagcgcaatcctcaggcattctggctgaggagattgttccggttgtgttgaaaaacaagaaaggtgttgtaacagaaatacaacatgatgagcatctgcgcccggaaacgacgctggaacagttacgtgggttaaaagcaccatttcgtgccaatggggtgattaccgcaggcaatgcttccggggtgaatgacggagccgctgcgttgattattgccagtgaacagatggcagcagcgcaaggactgacaccgcgggcgcgtatcgtagccatggcaaccgccggggtggaaccgcgcctgatggggcttggtccggtgcctgcaactcgccgggtgctggaacgcgcagggctgagtattcacgatatggacgtgattgaactgaacgaagcgttcgcggcccaggcgttgggtgtactacgcgaattggggctgcctgatgatgccccacatgttaaccccaacggaggcgctatcgccttaggccatccgttgggaatgagtggtgcccgcctggcactggctgccagccatgagctgcatcggcgtaacggtcgttacgcattgtgcaccatgtgcatcggtgtcggtcagggcatcgccatgattctggagcgtgtttga beta-atgaatgaaccgacccacgccgatgccttgatcatcgacgccgtgcgcacgcccattg ketothiolasegccgctatgccggggccctgagcagcgtgcgcgccgacgacctggcggccatcccg AAN68887.1ctcaaagccttgatccagcgtcaccccgaactggactggaaagccattgatgacgttatcttcggctgtgccaaccaggctggcgaagacaaccgcaacgtggcccacatggcgagcctgctggccgggctgccactcgaagtaccagggaccacgatcaaccgcctgtgcggttccggtctggatgccatcggtaatgcggcacgtgccctgcgctgcggtgaagcggggctcatgctggccggtggtgtggagtccatgtcgcgtgcaccgtttgtgatgggtaagtcggagcaggcattcgggcgtgcggccgagctgttcgacaccaccatcggctggcgtttcgtcaacccgctgatgaaggccgcctacggcatcgattcgatgccggaaacggctgaaaacgtggccgaacagttcggcatctcgcgcgccgaccaggatgcctttgccctgcgcagccagcacaaagccgcagcagctcaggcccgcggccgcctggcgcgggaaatcgtgccggtcgaaatcccgcaacgcaaaggcccagccaaagtggtcgagcatgacgagcacccgcgcggcgacacgaccctggagcagctggctcggctcgggacgccgtttcgtgaaggcggcagcgtaacggcgggtaatgcctccggcgtgaatgacggcgcttgcgccctgctgctggccagcagcgccgcggcccgccgccatgggttgaaggcccgcggccgcatcgtcggcatggcggtggccggggttgagcccaggctgatgggcattggtccggtgcctgcgacccgcaaggtgctggcgctcaccggcctggcactggctgacctggatgtcatcgaactcaatgaggcctttgccgcccaagggctggccgtgttgcgcgagctgggcctggccgacgacgacccgcgagtcaaccgcaacggcggcgccatcgccctgggccatcccctgggcatgagcggtgcccggttggtgaccactgccttgcacgagcttgaagaaacggccggccgctacgccctgtgcaccatgtgcatcggcgtaggccaaggcattgccatgatcatcgagcgcctctga beta-atgaaagaagttgtaatagctagtgcagtaagaacagcgattggatcttatggaaagtctketothiolase cttaaggatgtaccagcagtagatttaggagctacagctataaaggaagcagttaaaaaNP_349476.1 agcaggaataaaaccagaggatgttaatgaagtcattttaggaaatgttcttcaagcaggtttaggacagaatccagcaagacaggcatcttttaaagcaggattaccagttgaaattccagctatgactattaataaggtttgtggttcaggacttagaacagttagcttagcagcacaaattataaaagcaggagatgctgacgtaataatagcaggtggtatggaaaatatgtctagagctccttacttagcgaataacgctagatggggatatagaatgggaaacgctaaatttgttgatgaaatgatcactgacggattgtgggatgcatttaatgattaccacatgggaataacagcagaaaacatagctgagagatggaacatttcaagagaagaacaagatgagtttgctcttgcatcacaaaaaaaagctgaagaagctataaaatcaggtcaatttaaagatgaaatagttcctgtagtaattaaaggcagaaagggagaaactgtagttgatacagatgagcaccctagatttggatcaactatagaaggacttgcaaaattaaaacctgccttcaaaaaagatggaacagttacagctggtaatgcatcaggattaaatgactgtgcagcagtacttgtaatcatgagtgcagaaaaagctaaagagcttggagtaaaaccacttgctaagatagtttcttatggttcagcaggagttgacccagcaataatgggatatggacctttctatgcaacaaaagcagctattgaaaaagcaggttggacagttgatgaattagatttaatagaatcaaatgaagcttttgcagctcaaagtttagcagtagcaaaagatttaaaatttgatatgaataaagtaaatgtaaatggaggagctattgcccttggtcatccaatggagcatcaggtgcaagaatactcgttactcttgtacacgcaatgcaaaaaagagatgcaaaaaaaggcttagcaactttatgtataggtggcggacaaggaacagcaatattgctagaaaagtgctag beta-atgagagatgtagtaatagtaagtgctgtaagaactgcaataggagcatatggaaaaac ketothiolaseattaaaggatgtacctgcaacagagttaggagctatagtaataaaggaagctgtaagaa NP_149242.1gagctaatataaatccaaatgagattaatgaagttatttttggaaatgtacttcaagctggattaggccaaaacccagcaagacaagcagcagtaaaagcaggattacctttagaaacacctgcgtttacaatcaataaggtttgtggttcaggtttaagatctataagtttagcagctcaaattataaaagctggagatgctgataccattgtagtaggtggtatggaaaatatgtctagatcaccatatttgattaacaatcagagatggggtcaaagaatgggagatagtgaattagttgatgaaatgataaaggatggtttgtgggatgcatttaatggatatcatatgggagtaactgcagaaaatattgcagaacaatggaatataacaagagaagagcaagatgaattttcacttatgtcacaacaaaaagctgaaaaagccattaaaaatggagaatttaaggatgaaatagttcctgtattaataaagactaaaaaaggtgaaatagtctttgatcaagatgaatttcctagattcggaaacactattgaagcattaagaaaacttaaacctattttcaaggaaaatggtactgttacagcaggtaatgcatccggattaaatgatggagctgcagcactagtaataatgagcgctgataaagctaacgctctcggaataaaaccacttgctaagattacttcttacggatcatatggggtagatccatcaataatgggatatggagctttttatgcaactaaagctgccttagataaaattaatttaaaacctgaagacttagatttaattgaagctaacgaggcatatgcttctcaaagtatagcagtaactagagatttaaatttagatatgagtaaagttaatgttaatggtggagctatagcacttggacatccaataggtgcatctggtgcacgtattttagtaacattactatacgctatgcaaaaaagagattcaaaaaaaggtcttgctactctatgtattggtggaggtcagggaacagctctcgtagttgaaagagactaa 3-oxoadipyl-atgttcaagaaatcagctaatgatattgttgttattgcagcaaagagaactccaatcaccaCoA thiolaseagtcaattaaaggtgggttgagtagattatttcctgaggaaatattatatcaagtggttaagggtactgtatcagattcacaagttgatttaaacttgattgatgatgtgttagtcggtacggtcttgcaaactttagggggacagaaagctagtgccttggccattaaaaagattggattcccaattaagaccacggttaatacggtcaatcgtcaatgtgctagttctgctcaagcgattacttatcaagcaggtagtttgcgtagtggggagaatcaatttgctattgctgctggagtagaaagtatgactcatgattattttcctcatcgtgggattcccacaagaatttctgaatcatttttagctgatgcatccgatgaagctaaaaacgtcttgatgccaatggggataaccagtgaaaatgttgccactaaatatggaatttctcgtaaacaacaagatgagtttgcccttaattctcatttgaaagcagacaaggctacaaaactgggtcattttgcaaaagaaatcattcctattcaaacaacggatgaaaacaaccaacacgtttcaataaccaaagatgatggtataaggggaagttcaacaattgaaaagttgggtggcttaaaacctgtgttcaaggatgatgggactactactgctggtaattcctcgcaaatttcagatggagggtctgctgtgattttaactactcgtcaaaatgctgagaaatcgggagtaaagccaatagctagatttattggttcgtcagtagctggtgttccttcgggacttatgggaattggtccatcggctgctattcctcaattgttgtcgagattaaatgttgacacgaaagacattgatatttttgaattgaacgaggcatttgcatcccaactgatttattgtattgaaaaattgggtcttgattatgataaagtcaatccatatggtggagctatagccttgggacatccattaggagccactggcgcaagagttacggcaacgttgcttaatggattaaaagatcagaataaagagttgggtgtcatctcaatgtgcacatccacaggtcaaggatacgctgccttgtttgctaacgagtag 3-oxoadipyl-atggatagattaaatcaattaagtggtcaattaaaaccaacttcaaaacaatcccttactcaCoA thiolaseaaagaacccagacgatgttgtcatcgttgcagcatacagaactgccatcggtaaaggtttcaaagggtctttcaaatctgtgcaatctgaattcatcttgactgaattcttgaaagaatttattaaaaagactggagtcgatgcatctttgattgaagatgttgctattggtaacgttttgaaccaagctgctggtgccaccgaacacagaggtgctagtttggctgcaggtattccttacactgcagctttccttgccatcaacagattgtgttcctcagggttaatggccatttctgacattgccaacaaaatcaaaaccggtgaaatcgaatgtggtcttgctggtggtattgaatccatgtctaaaaactatggtagtccaaaagttattccaaagattgacccacacttggctgatgacgaacaaatgagtaaatgtttgattccaatgggtatcaccaacgaaaatgttgctaatgaattcaacattccaagagaaaaacaagatgcctttgctgctaaatcttatagtaaagccgaaaaagccatctcctctggagctttcaaagatgaaatcttaccaatcagatccattatcagatccccagacggttctgaaaaagaaatcattgtcgataccgacgaaggtccaagaaagggtgttgacgctgcttccttgagcaaattgaaaccagcatttggtggtactaccactgccggtaacgcttctcaaatttcagatggtgctgctggtgttttattgatgaagagaagtttggctgaagccaaaggttacccaattgttgctaaatacattgcttgttcaactgttggtgttccgccagaaatcatgggtgttggtccagcttacgccattccagaagtgttgaagagaactggattgactgtggatgacgttgatgtgtttgaaatcaacgaagcttttgctgctcaatgtctttactcagctgaacaatgtaatgttccagaagaaaaattgaacataaacggtggtgccatcgctttaggtcatcctcttggttgtactggtgccagacaatatgccactatcttgagattgttgaaaccaggtgaaattggtttgacttctatgtgtatcggtagtggtatgggtgctgcctccatattgattaaggaat ag3-oxoadipyl-atgtcatccaaacaacaatacttgaagaagaatcctgacgatgtcgttgtcgttgcagcatCoA thiolaseacagaactgctttaaccaaaggtggaagaggtggattcaaagatgttggatctgatttccttttgaaaaaattgactgaagaatttgttaaaaaaactggtgttgaccctaaaatcattcaagatgctgccattggtaatgtcttgaacagaagagctggtgatttcgaacatagaggtgcattattatctgctggattaccttattcagttccatttgttgcccttaacagacaatgttcatctgggttaatggccatttctcaagtggccaacaagatcaagactggtgaaattgaatgtggtttagctggtggtgttgaaagtatgacaaaaaactatggtccagaagcattgattgctattgaccctgcttatgaaaaagacccagaatttgttaaaaacggtattccaatgggtattactaatgaaaatgtttgtgccaaattcaatatttcaagagatgttcaagatcaatttgctgctgaatcttatcaaaaagctgaaaaggcacaaaaagaaggtaaatttgatgatgaaattttaccaattgaagttttccaagaagatgaagatgctgaagatgaagacgaagatgaagatgaagatgctgaaccaaaagaaaaattggttgttattagtaaagatgaaggtattagaccaggtgttactaaagaaaaattggctaaaattaaaccagctttcaaatctgatggtgtatcttcagctggtaactcttcacaagtttccgatggtgctgccttggtgttattgatgaaacgttcatttgctgaaaagaatggattcaaaccattggctaaatacatttcttgtggtgttgctggtgtcccaccagaaattatgggtattggtccagctgttgccattccaaaagttttgaaacaaactggattatcagtcagtgatattgatatttatgaaatcaatgaagcatttgccggtcaatgtttgtactcaattgaaagttgtaatattccaagagaaaaagtcaatcttaatgggggtgctattgccttgggtcaccctcttggttgtactggtgctagacaatacgctactattttaagattgttaaaaccaggtgaatttggtgtgacttctatgtgtattggtactggtatgggtgctgcttctgttttggttagagaa taabeta- atgagccgcgaggtattcatctgcgatgccgtgcgcacgccgatcggccgtttcggcgketoadipyl gcagtctttccgcggtgcgcgccgacgacctcgcggcggtgccgctgaaggccctggCoA thiolase tcgagcgcaacccgggggtcgactggtcggcgttggacgaggtgttcctcggctgcgpcaF ccaaccaggccggcgaggacaaccgtaacgtggcgcgcatggcgctgctgctggccggtttgccggagagcgtgcccggcgtcaccctcaaccgcctctgcgcctcggggatggacgccatcggcacggcgttccgcgccatcgcctgcggcgagatggagctggccatcgccggcggcgtcgagtcgatgtcgcgcgcgccgtacgtgatgggcaaggccgatagcgccttcggtcgcggccagaagatcgaggacaccaccatcggctggcgcttcgtcaatccgctgatgaaggagcagtacggcatcgacccgatgccgcagaccgccgacaacgtcgccgacgactatcgcgtgtcgcgtgccgaccaggatgccttcgccctgcgcagccagcagcgcgccggcagggcgcaggaggccggtttcttcgccgaggaaatcgtcccggtgacgattcgcgggcgcaagggcgacaccctggtcgagcacgacgagcatccgcgtcccgacaccaccctggaggcgctggcccggctcaagccggtcaacgggccggagaagaccgtcaccgccggcaacgcgtccggggtcaacgacggcgccgccgcgctggtcctggcctccgccgaggcagtggagaagcacggcctgactccgcgcgcgcgggtgctgggcatggccagcgccggcgtcgccccacggatcatgggcatcggcccggtgccggcggtgcgcaagctgctgcggcgcctggacctggcgatcgacgccttcgacgtgatcgaactcaacgaagccttcgccagccagggcctggcctgcctgcgcgaactgggcgtggccgacgacagtgagaaggtcaacccgaacggcggtgccatcgccctcggccacccgctggggatgagcggtgcgcggctggtcctcaccgcgctccatcaacttgagaagagcggcggccggcgcggcctggcgaccatgtgcgtaggcgtcggccaaggcctggcgctggccatcgagcgggtctga acyl-CoAatgctcgatgcctatatctacgccggcctgcgtacgcctttcggccggcatgccggtgc thiolaseactctcgacggtgcgtccggacgacctggccggcctgctgctggcgcgtctcgcggaaacctccgggttcgccgtcgacgacctggaggatgtgatcctcggttgcaccaaccaggccggcgaagacagccgcaacctggcgcgcaacgcgctgctcgcagccggcctgccggcgcggctgcccgggcagacggtcaaccgcttgtgtgccagcggactgtcggcggtgatcgacgcggcgcgcgcgatcagttgcggtgagggccggctgtacctggccggcggcgccgaaagcatgtcccgggcgccgttcgtcatgggcaaggcggagagcgccttcagccgcacgctggaggtcttcgacagcaccatcggcgcgcgcttcgccaaccccaggctggtcgagcgctatggcaacgacagcatgccggagaccggcgacaacgtggcccgcgccttcggcatcgcccgcgaagacgccgaccgtttcgccgcttcttcccaggcgcgctaccaggctgcgctggaggagggctttttcctcggcgagatccttccggtggaggtgcgtgccggacgcaagggcgagacgcggctggtggagcgcgacgagcatccgcgaccgcaggccgacctggcggccctggcgcgcttgccggcgttgttcgccggtggggtagtgaccgccggtaatgcgtctgggatcaacgacggggcggcggtagtgctgctgggcgatcgcgcgatcggcgagcgcgagggcatccggccgttggcgcggatcctcgccagcgccagcgtcggcgtcgagccccggttgatgggcatcggcccgcagcaggcgatcctccgcgcgctgcaacgcgccggcatcgacctggacgaggtcggcctgatcgagatcaacgaagccttcgcgccgcaggtcctggcctgcctgaagttgctcggcctggactacgaggacccgcgggtcaatccccatggcggcgccattgccctcggccatccgctcggcgcctccggtgcgcgcctggtgctcaccgccgcccgcgggctgcaacgcatcgagcggcgctacgcggtggtcagcctgtgcgtcgggctcggccagggcgtggcgatggtgatcgagcgctgccgatga 3-oxoadipyl-atgcacgacgtattcatctgtgacgccatccgtaccccgatcggccgcttcggcggcgc CoA thiolasecctggccagcgtgcgggccgacgacctggccgccgtgccgctgaaggcgctgatcgagcgcaaccctggcgtgcagtgggaccaggtagacgaagtgttcttcggctgcgccaaccaggccggtgaagacaaccgcaacgtggcccgcatggcactgctgctggccggcctgccggaaagcatcccgggcgtcaccctgaaccgtctgtgcgcgtcgggcatggatgccgtcggcaccgcgttccgcgccatcgccagcggcgagatggagctggtgattgccggtggcgtcgagtcgatgtcgcgcgccccgttcgtcatgggcaaggctgaaagcgcctattcgcgcaacatgaagctggaagacaccaccattggctggcgtttcatcaacccgctgatgaagagccagtacggtgtggattccatgccggaaaccgccgacaacgtggccgacgactatcaggtttcgcgtgctgatcaggacgctttcgccctgcgcagccagcagaaggctgccgctgcgcaggctgccggcttctttgccgaagaaatcgtgccggtgcgtatcgctcacaagaagggcgaaatcatcgtcgaacgtgacgaacacctgcgcccggaaaccacgctggaggcgctgaccaagctcaaaccggtcaacggcccggacaagacggtcaccgccggcaacgcctcgggcgtgaacgacggtgctgcggcgatgatcctggcctcggccgcagcggtgaagaaacacggcctgactccgcgtgcccgcgttctgggcatggccagcggcggcgttgcgccacgtgtcatgggcattggcccggtgccggcggtgcgcaaactgaccgagcgtctggggatagcggtaagtgatttcgacgtgatcgagcttaacgaagcgtttgccagccaaggcctggcggtgctgcgtgagctgggtgtggctgacgatgcgccccaggtaaaccctaatggcggtgccattgccctgggccaccccctgggcatgagcggtgcacgcctggtactgactgcgttgcaccagctggagaagagtggcggtcgcaagggcctggcgaccatgtgtgtgggtgtcggccaaggtctggcgttggccatcgagcgggtttg a3-oxoadipyl- atgaccgacgcctacatctgcgatgcgattcgcacacccatcggccgctacggcggcCoA thiolase gccctgaaagacgttcgtgccgacgatctcggcgcggtgccgctcaaggcgctgatcgaacgcaaccggaacgtcgactggtcggcgatcgacgacgtgatctatggctgcgcgaaccaggccggcgaagacaaccgcaacgtcgcgcgcatgtccgcgctgctcgcgggcttgccgaccgccgtgccgggcacgacgctgaaccggttatgcggctcgggcatggacgccgtcggcacggccgcgcgcgcgatcaaggcgggcgaggcacgcttgatgatcgcgggcggcgtcgaaagcatgacgcgcgcgccgttcgtgatgggcaaggccgccagcgcattcgcgcgccaggctgcgattttcgacacgacgatcggctggcgtttcattaatccgctgatgaaacagcaatacggcgtcgattcgatgcccgagacggccgagaacgtcgcggtcgactacaacatcagccgcgccgaccaggatctattcgcgctgcgcagccagcagaaggccgcgcgtgcgcagcaggacggcacgctcgccgccgaaatcgtccccgtcacgattgcgcagaaaaaaggcgacgcgctcgtcgtatcgctcgacgagcatccgcgcgaaacatcgctcgaagcgctcgcgaagctgaagggcgtcgtgcgtcccgacggctcggtcacggccggcaacgcgtcaggcgtcaacgacggcgcatgcgcactgctgctcgccaacgcggaagccgccgatcaatatgggctgcgccgccgcgcgcgtgtcgtcggcatggcgagcgccggcgtcgagccgcgcgtgatgggtatcggcccggcgccggccacgcagaaactgttgcgccagctcggcatgacgatcgaccagttcgacgtgatcgagctgaacgaagcgttcgcgtcgcagggtctcgcggtgctgcgcatgctcggtgtcgccgacgacgatccgcgcgtgaaccccaacggcggtgcgatcgcgctcggccatccgctcggcgcatcgggtgcgcggctcgtgaccacggcgcttcaccaactcgagcgtacgggcggccgctttgcgctctgtacgatgtgcatcggcgtcggccagggcatcgcgatcgcgatc gaacgcgtgtaabeta- gtgatggccacctcaagacttgtctgcagcaatttaacgaagcaatgctttacgatctcgtketothiolase cacgtgctgctagccaatttaccgatgtggtattcgtgggtgccgcacgaacaccggtcggatcgtttcgctcttcgctttccactgttccagccactgtcctcggagctgaggctattaagggtgcacttaaacatgccaatctaaaaccctcacaagtgcaagaggtgttctttggctgtgtcgttccatccaactgtggacaagttcctgcccgtcaagcgacacttggagctggatgcgatccttcgacaatcgttacaactctcaataaattgtgcgcctcgggaatgaagtcgattgcttgtgccgcctcacttttgcaacttggtcttcaagaggttaccgttggtggcggtatggagagcatgagcttagtgccgtactatcttgaacgtggtgaaactacttatggtggaatgaagctcatcgacggtatcccaagagatggtccgactgatgcatatagtaatcaacttatgggtgcatgcgctgataatgtggctaaacgattcaacatcacccgtgaggaacaggataaattcgctattgaaagctataaacgatctgctgctgcatgggagagtggagcatgcaaagctgaagtagttcctattgaagtgacaaagggcaagaaaacatacattgtcaacaaggatgaggaatacatcaaagtcaacttcgagaagcttcccaaactgaaacccgccttcttgaaagacggaaccatcacggctggcaatgcttcaacactgaacgatggtgctgcggcagttgtgatgacgactgtcgaaggagcgaaaaaatacggtgtgaaaccattggcccgattgctctcatatggtgatgcggcaacaaatccagtcgattttgctattgcaccatcaatggttatcccaaaggtacttaaattggctaatctcgagatcaaggatattgatttgtgggaaatcaacgaggctttcgccgttgttccccttcattcaatgaagacactcggtatcgatcactcgaaagtgaacattcatggtggtggcgtatctcttggacatcctattggaatgtctggagctcgaattatcgttcatctgattcatgcgttgaaacctggccagaaaggctgcgctgcaatctgcaatggtggcggtggcgctggtggaatggtcatcgagaaattgtaa

The genes were expressed in E. coli and the proteins purified usingNi-NTA spin columns and quantified. To assay enzyme activity in vitro, a5× CoA:DTNB (Ellman's reagent or 5,5′-dithiobis-(2-nitrobenzoic acid))mixture was prepared. The mixture consisted of 10 mM succinyl-CoA, 5 mMacetyl-CoA, 30 mM DTNB in 100 mM Tris buffer, pH 7.4. Five μL of theCoA:DTNB mixture was added to 0.5 μM purified thiolase enzyme in 100 mMTris buffer, pH 7.8 in a final volume of 50 μL. The reaction wasincubated at 30° C. for 30 minutes, then quenched with 2.5 μL 10% formicacid and samples frozen at −20° C. until ready for analysis by LC/MS.Because many thiolases can condense two acetyl-CoA molecules intoacetoaceytl-CoA, production of acetoacetyl-CoA was examined. FIG. 6shows that 3 thiolases demonstrated thiolase activity which resulted inacetoacetyl-CoA formation. These were fadAx from Pseudomonas putida,thiA from Clostridium acetobutylicum and thiB also from Clostridiumacetobutylicum. When enzyme assays were examined for condensation ofsuccinyl-CoA and acetyl-CoA into β-ketoadipyl-CoA, several enzymesdemonstrated the desired activity; paaJ from Escherichia coli (Nogaleset al., Microbiol. 153:357-365 (2007)), phaD from Pseudomonas putida(Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)), bktfrom Burkholderia ambifaria AMMD, pcaF from Pseudomonas putida KT2440(Harwood et al., J. Bacteriol. 176:6479-6488 (1994)), and pcaF fromPseudomonas aeruginosa PAO1. There was excellent specificity between thethiolases. Those that generated significant amounts of β-ketoadipyl-CoAdid not produce significant amounts of acetoacetyl-CoA and likewisethose that made acetoacetyl-CoA did not make detectable amounts ofβ-ketoadipyl-CoA.

EXAMPLE II Preparation of Terepthalate from Acetylene and Muconate

This Example provides conditions for the thermal inverse electron demandDiels-Alder reaction for the preparation of PTA from acetylene andmuconate.

A lab-scale Parr reactor is flushed with nitrogen gas, evacuated andcharged with (1 equivalent) trans, trans-muconic acid and (10equivalents) acetylene. The reactor is then heated to 200° C. and heldat this temperature for 12 hours. An initial pressure of 500 p.s.i.g. isapplied. The reactor is then vented, exposed to air and cooled. Thecontents of the reactor are distilled at room temperature and pressureto yield volatile and nonvolatile fractions. The contents of eachfraction are evaluated qualitatively by gas chromatographic analysis(GC-MS).

For quantitative analysis, standards of the starting materials and theexpected products, cyclohexa-2,5-diene-1,4-dicarboxylate andterepthalate, are prepared. A known amount of cyclohexane is mixed witha known amount of the volatile fraction and the mixture is subjected togas chromatography. The cyclohexane and terepthalate components arecondensed from the effluent of the chromatogram into a single trap, thecontents of which are diluted with carbon tetrachloride or CDCl₃ andthen examined by NMR spectroscopy. Comparison of the appropriate areasof the NMR spectrum permits calculation of yields.

EXAMPLE III Preparation of a Muconate Producing Microbial Organism, inwhich the Muconate is Derived from Succinyl-CoA

This example describes the generation of a microbial organism that hasbeen engineered to produce muconate from succinyl-CoA and acetyl-CoA viabeta-ketoadipate, as shown in FIG. 2. This example also provides amethod for engineering a strain that overproduces muconate.

Escherichia coli is used as a target organism to engineer amuconate-producing pathway as shown in FIG. 5. E. coli provides a goodhost for generating a non-naturally occurring microorganism capable ofproducing muconate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic, microaerobic or aerobic conditions.

First, an E. coli strain is engineered to produce muconate fromsuccinyl-CoA via the route outlined in FIG. 2. For the first stage ofpathway construction, genes encoding enzymes to transform centralmetabolites succinyl-CoA and acetyl-CoA to 2-maleylacetate (FIG. 2, StepA) is assembled onto vectors. In particular, the genes pcaF (AAA85138),pcaIJ (AAN69545 and NP_746082) and clcE (O30847) genes encodingbeta-ketothiolase, beta-ketoadipyl-CoA transferase and 2-maleylacetatereductase, respectively, are cloned into the pZE13 vector (Expressys,Ruelzheim, Germany), under the control of the PA1/lacO promoter. Thegenes bdh (AAA58352.1) and fumC (P05042.1), encoding 2-maleylacetatedehydrogenase and 3-hydroxy-4-hexenedioate dehydratase, respectively,are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) underthe PA1/lacO promoter. The two sets of plasmids are transformed into E.coli strain MG1655 to express the proteins and enzymes required formuconate synthesis from succinyl-CoA.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of muconatepathway genes is corroborated using methods well known in the art fordetermining polypeptide expression or enzymatic activity, including forexample, Northern blots, PCR amplification of mRNA and immunoblotting.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individually activities. The ability of the engineeredE. coli strain to produce muconate through this pathway is confirmedusing HPLC, gas chromatography-mass spectrometry (GCMS) or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional muconate synthesispathway from succinyl-CoA are further augmented by optimization forefficient utilization of the pathway. Briefly, the engineered strain isassessed to determine whether any of the exogenous genes are expressedat a rate limiting level. Expression is increased for any enzymesexpressed at low levels that can limit the flux through the pathway by,for example, introduction of additional gene copy numbers.

After successful demonstration of enhanced muconate production via theactivities of the exogenous enzymes, the genes encoding these enzymesare inserted into the chromosome of a wild type E. coli host usingmethods known in the art. Such methods include, for example, sequentialsingle crossover (Gay et al., J. Bacteriol. 153:1424-1431 (1983)) andRed/ET methods from GeneBridges (Zhang et al., Improved RecT or RecETcloning and subcloning method (WO/2003/010322)). Chromosomal insertionprovides several advantages over a plasmid-based system, includinggreater stability and the ability to co-localize expression of pathwaygenes.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of muconate. One modeling method isthe bilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of muconate.Adaptive evolution also can be used to generate better producers of, forexample, the 4-acetylbutyrate intermediate or the muconate product.Adaptive evolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the muconate producer to further increaseproduction.

For large-scale production of muconate, the above muconatepathway-containing organism is cultured in a fermenter using a mediumknown in the art to support growth of the organism under anaerobicconditions. Fermentations are performed in either a batch, fed-batch orcontinuous manner. Anaerobic conditions are maintained by first spargingthe medium with nitrogen and then sealing culture vessel (e.g., flaskscan be sealed with a septum and crimp-cap). Microaerobic conditions alsocan be utilized by providing a small hole for limited aeration. The pHof the medium is maintained at a pH of 7 by addition of an acid, such asH₂SO₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm), and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu) with an HPX-087 column (BioRad), using a refractive indexdetector for glucose and alcohols, and a UV detector for organic acids,Lin et al., Biotechnol. Bioeng., 775-779 (2005).

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

What is claimed is:
 1. A transformed Escherichia coli having a muconatepathway that produces muconate pathway enzymes, said muconate pathwayenzymes comprising a (1) beta-ketothiolase, (2) an enzyme selected frombeta-ketoadipyl-CoA hydrolase and beta-ketoadipyl-CoA transferase, (3)beta-ketoadipate enol-lactone hydrolase, (4) muconolactone isomerase,(5) muconate cycloisomerase, and (6) muconate cis/trans isomerase,wherein said Escherichia coli comprises at least one exogenous nucleicacid encoding a muconate pathway enzyme obtained from bacteria, yeast,fungi, plant or mammal, and wherein the at least one exogenous nucleicacid is overexpressed resulting in increased synthesis or accumulationof a muconate.
 2. The Escherichia coli organism of claim 1, wherein saidEscherichia coli organism comprises two exogenous nucleic acids eachencoding a muconate pathway enzyme.
 3. The Escherichia coli organism ofclaim 1, wherein said Escherichia coli organism comprises threeexogenous nucleic acids each encoding a muconate pathway enzyme.
 4. TheEscherichia coli organism of claim 1, wherein said Escherichia coliorganism comprises four exogenous nucleic acids each encoding a muconatepathway enzyme.
 5. The Escherichia coli organism of claim 1, whereinsaid Escherichia coli organism comprises five exogenous nucleic acidseach encoding a muconate pathway enzyme.
 6. The Escherichia coliorganism of claim 1, wherein said Escherichia coli organism comprisessix exogenous nucleic acids each encoding a muconate pathway enzyme. 7.The Escherichia coli organism of claim 1, wherein said at least oneexogenous nucleic acid is a heterologous nucleic acid.
 8. TheEscherichia coli organism of claim 1, wherein said Escherichia coliorganism is in a substantially anaerobic culture medium.
 9. A method forproducing muconate, comprising culturing the Escherichia coli organismaccording to any one of claim 1 and claims 2-8.
 10. The method of claim9, further comprising a starting material selected from pyruvate,succinic semialdehyde, and lysine.
 11. The Escherichia coli organism ofclaim 1, wherein the at least one exogenous nucleic acid encoding amuconate pathway enzyme has a mitochondrial leader sequence removed. 12.The method of claim 9, wherein the culturing said Escherichia coliorganism is in a substantially anaerobic culture medium.
 13. The methodof claim 9, wherein a starting material is selected from pyruvate,succinic semialdehyde, and lysine.